Recombinant factor viii having enhanced stability following mutation at the a1-c2 domain interface

ABSTRACT

The invention relates to a recombinant factor VIII that includes one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII. The one or more mutations include substitution of one or more amino acid residues with either a cysteine or an amino acid residue having a higher hydrophobicity. This results in enhanced stability of factor VIII. Methods for making the recombinant factor VIII, pharmaceutical compositions containing the recombinant factor VIII, and use of the recombinant factor VIII for treating hemophilia A are also disclosed.

This application is a division of U.S. patent application Ser. No.13/231,948, filed Sep. 13, 2011, which claims the priority benefit ofU.S. Provisional Patent Application Ser. No. 61/382,919, filed Sep. 14,2010, which are hereby incorporated by reference in their entirety.

This invention was made with government support under grant numberHL38199 and HL76213 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Hemophilia A, the most common of the severe, inherited bleedingdisorders, results from a deficiency or defect in the plasma proteinfactor VIII. There is no cure for Hemophilia A and treatment consists ofreplacement therapy using preparations of (purified) plasma or therecombinant protein.

Factor VIII circulates as a non-covalent, metal ion-dependentheterodimer. This procofactor form of the protein contains a heavy chain(HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprisedof (a3)A3C1C2 domains, with the lower case a representing short (˜30-40residue) segments rich in acidic residues (see Fay, “Activation ofFactor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15(2004)). Factor VIII is activated by proteolytic cleavages at the A1A2,A2B and A3A3 junctions catalyzed by thrombin or factor Xa. The productof this reaction, factor VIIIa, is a heterotrimer comprised of subunitsdesignated A1, A2, and A3C1C2 that functions as a cofactor for theserine protease factor IXa in the membrane-dependent conversion ofzymogen factor X to the serine protease, factor Xa (see Fay, “Activationof Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15(2004)).

Reconstitution studies have shown that the factor VIII heterodimericstructure is supported by both electrostatic and hydrophobicinteractions (Fay, “Reconstitution of Human Factor VIII from IsolatedSubunits,” Arch Biochem Biophys. 262:525-531 (1988); Ansong et al.,“Factor VIII A1 Domain Residues 97-105 Represent a LightChain-interactive Site,” Biochemistry 45:13140-13149 (2006)), and theinter-chain affinity is further strengthened by factor VIII binding vonWillebrand factor (Fay, “Reconstitution of Human Factor VIII fromIsolated Subunits,” Arch Biochem Biophys. 262:525-531 (1988); Kaufman etal., “Regulation of Factor VIII Expression and Activity by vonWillebrand Factor,” Thromb Haemost. 82:201-208 (1999)). Metal ions alsocontribute to the inter-chain affinity and activity parameters(Wakabayashi et al., “Metal Ion-independent Association of Factor VIIISubunits and the Roles of Calcium and Copper Ions for Cofactor Activityand Inter-subunit Affinity,” Biochemistry 40:10293-10300 (2001)).Calcium is required to yield the active factor VIII conformation.Mutagenesis studies mapped a calcium-binding site to a segment rich inacidic residues within the A1 domain (residues 110-126) and identifiedspecific residues within this region prominent in the coordination ofthe ion (Wakabayashi et al., “Residues 110-126 in the A1 Domain ofFactor VIII Contain a Ca²⁺ Binding Site Required for Cofactor Activity,”J Biol. Chem. 279:12677-12684 (2004)). A recent intermediate resolutionX-ray structure (Shen et al., “The Tertiary Structure and DomainOrganization of Coagulation Factor VIII,” Blood 111:1240-1247 (2008))confirmed this calcium-binding site as well as suggested a secondpotential site within the A2 domain. This structure also showedoccupancy of the two type 1 copper ion sites within the A1 and A3domains. Earlier functional studies have shown that copper ionsfacilitate the association of the heavy and light chains to form theheterodimer, increasing the inter-chain affinity by several-fold atphysiologic pH (Fay et al., “Human Factor VIIIa Subunit Structure:Reconstruction of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer andA2 Subunit,” J Biol. Chem. 266:8957-8962 (1991); Wakabayashi et al.,“pH-dependent Association of Factor VIII Chains: Enhancement of Affinityat Physiological pH by Cu²⁺ ,” Biochim Biophys Acta. 1764:1094-1101(2006); Ansong et al., “Factor VIII A3 Domain Residues 1954-1961Represent an A1 Domain-Interactive Site,” Biochemistry 44:8850-8857(2005)).

The instability of factor VIIIa results from weak electrostaticinteractions between the A2 subunit and the A1/A3C1C2 dimer (Fay et al.,“Human Factor VIIIa Subunit Structure: Reconstruction of Factor VIIIafrom the Isolated A1/A3-C1-C2 Dimer and A2 Subunit,” J Biol. Chem.266:8957-8962 (1991); Lollar et al., “pH-dependent Denaturation ofThrombin-activated Porcine Factor VIII,” J Biol. Chem. 265:1688-1692(1990)) and leads to dampening of factor Xase activity (Lollar et al.,“Coagulant Properties of Hybrid Human/Porcine Factor VIII Molecules,” JBiol Chem. 267:23652-23657 (1992); Fay et al., “Model for the FactorVIIIa-dependent Decay of the Intrinsic Factor Xase: Role of SubunitDissociation and Factor IXa-catalyzed Proteolysis,” J Biol. Chem.271:6027-6032 (1996)). Limited information is available regarding theassociation of the A2 subunit in factor VIIIa, and residues in both theA1 and A3 domains appear to make contributions to the retention of thissubunit. Several factor VIII point mutations have been shown tofacilitate the dissociation of A2 relative to WT and these residueslocalize to either the A1-A2 domain interface (Pipe et al., “MildHemophilia A Caused by Increased Rate of Factor VIII A2 SubunitDissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIain vivo,” Blood 93:176-183 (1999); Pipe et al., “Hemophilia A MutationsAssociated with 1-stage/2-stage Activity Discrepancy DisruptProtein-protein Interactions within the Triplicated A Domains ofThrombin-activated Factor VIIIa,” Blood 97:685-691 (2001)) or the A2-A3domain interface (Hakeos et al., “Hemophilia A Mutations within theFactor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and CauseOne-stage/Two-stage Activity Discrepancy,” Thromb Haemost. 88:781-787(2002)). These factor VIII variants demonstrate a characteristicone-stage/two-stage assay discrepancy (Duncan et al., “FamilialDiscrepancy Between the One-stage and Two-stage Factor VIII Methods in aSubgroup of Patients with Haemophilia A,” Br J Haematol. 87:846-848(1994); Rudzki et al., “Mutations in a Subgroup of Patients with MildHaemophilia A and a Familial Discrepancy Between the One-stage andTwo-stage Factor VIII:C Methods,” Br J Haematol. 94:400-406 (1996)),with significant reductions in activity values determined by the latterassay as a result of increased rates of A2 subunit dissociation.

Significant interest exists in stabilizing factor VIIIa, since a morestable form of the protein would represent a superior therapeutic forhemophilia A, potentially requiring less material to treat the patient(Fay et al., “Mutating Factor VIII: Lessons from Structure to Function,”Blood Reviews 19:15-27 (2005)). To this end, preparations of factor VIIIhave been described where mutations have been made in the recombinantprotein to prevent the dissociation of the A2 subunit by introducingnovel covalent bonds between A2 and other factor VIII domains (Pipe etal., “Characterization of a Genetically EngineeredInactivation-resistant Coagulation Factor VIIIa,” Proc Natl Acad Sci USA94:11851-11856 (1997); Gale et al., “An Engineered Interdomain DisulfideBond Stabilizes Human Blood Coagulation Factor VIIIa,” J. Thromb.Haemostasis 1:1966-1971 (2003)). However, it has since been suggestedthat these types of mutation may not be desirable in a therapeuticfactor VIII, because they substantially eliminate means fordown-regulation. This situation could yield a prothrombotic condition,which may cause harm. Thus, it would be desirable to enhance thestability of both factor VIII and factor VIIIa, but in a manner thatminimizes the likelihood of promoting prothrombotic conditions.

In U.S. Patent Application Publ. No. 20090118184, a number ofrecombinant factor VIII proteins are identified that possess one or moremutations that result in enhanced stability of both factor VIII andfactor VIIIa. These recombinant factor VIII proteins have one or moresubstitutions of a charged amino acid residue with a hydrophobic aminoacid residue at either or both of the A1-A2 or A2-A3 domain interfaces.Despite the improvements made in the stability of recombinant factorVIII proteins (and their active forms, factor VIIIa), the need forfurther improvements continue to exist.

The intermediate resolution X-ray structures of factor VIII (Shen etal., “The Tertiary Structure and Domain Organization of CoagulationFactor VIII,” Blood 111:1240-1247 (2008); Ngo et al., “Crystal Structureof Human Factor VIII: Implications for the Formation of the FactorIXa:Factor VIIIa Complex,” Structure 16:597-606 (2008)) show closecontact between A1 (heavy chain) and C2 (light chain) domains. It wasrecently reported that a factor VIII variant lacking the C2 domainretained the capacity to bind phospholipid membranes, albeit with amarked reduction in affinity (Wakabayashi et al., “Factor VIII Lackingthe C2 Domain Retains Cofactor Activity in vitro,” J. Biol. Chem.285:25176-25184 (2010)), supporting a direct role for the C1 domain inthis interaction. Furthermore, deletion of the C2 domain did not grosslyalter a number of functional properties including the rate ofprocofactor activation by thrombin, affinity of factor VIIIa for factorIXa, K_(m) of factor Xase for substrate factor X, or k_(cat) for factorXa generation. However, this deletion did significantly destabilize thecofactor, as judged by increased rates of activity decay followingexposure to elevated temperature or chemical denaturants. While contactsbetween the A1 and C2 domains of factor VIII appear to contribute toprotein, and in particular heterodimer stability, little information isavailable on specific interactions and their functional significance. Itwould be desirable, therefore, to identify amino acid residues that canbe modified to enhance stability between the A1 (heavy chain) and C2(light chain) domains.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a recombinant factorVIII that includes one or more mutations at an interface of A1 and C2domains of recombinant factor VIII. This results in enhanced stabilityof factor VIII. The one or more mutations include substitution of one ormore amino acid residues with either a cysteine or an amino acid residuehaving a higher hydrophobicity.

According to one embodiment, the recombinant factor VIII includes one ormore mutations at an interface of A1 and C2 domains of recombinantfactor VIII that result in enhanced stability of factor VIII, whereinthe one or more mutations comprise substitution of one or more aminoacid residues with an amino acid residue having a higher hydrophobicity.

According to another embodiment, the recombinant factor VIII includes(i) two or more mutations at an interface of A1 and C2 domains ofrecombinant factor VIII, wherein the two or more mutations comprisesubstitution of two or more amino acid residues with a Cysteine residueto afford a disulfide bond between the A1 and C2 domains; and (ii) oneor more mutations at an interface of A1 and A2 domains or an interfaceof A2 and A3 domains of recombinant factor VIII, said one or moremutations comprising substitution of one or more charged amino acidresidues with a hydrophobic amino acid residue. The recombinant factorVIII possessing these mutations exhibits enhanced stability of bothfactor VIII and factor VIIIa.

A second aspect of the present invention relates to the recombinantfactor VIII according to the first aspect of the present invention,wherein the recombinant factor VIII further includes one or more of (i)factor IXa and/or factor X binding domains modified to enhance theaffinity of the recombinant factor VIII for one or both of factor IXaand factor X; (ii) modified sites that enhance secretion in culture;(iii) modified serum protein binding sites that enhance the circulatinghalf-life thereof; (iv) at least one glycosylation recognition sequencethat is effective in decreasing antigenicity and/or immunogenicitythereof; (v) a modified A1 domain calcium-binding site that improvesspecific activity of the recombinant factor VIIIa; (vi) modifiedactivated protein C-cleavage site; (vii) a modified A1 and A2 domaininterface; and (viii) a modified A2 and A3 domain interface.

A third aspect of the present invention relates to a pharmaceuticalcomposition that includes the recombinant factor VIII according to thepresent invention.

A fourth aspect of the present invention relates to an isolated nucleicacid molecule encoding a recombinant factor VIII of the presentinvention. Also included within this aspect of the present invention arerecombinant DNA expression systems that contain a DNA molecule encodingthe recombinant factor VIII of the present invention, and recombinanthost cells that contain the DNA molecule and/or recombinant expressionsystem.

A fifth aspect of the present invention relates to a method of making arecombinant factor VIII that includes: growing a host cell according tothe fourth aspect of the present invention under conditions whereby thehost cell expresses the recombinant factor VIII; and isolating therecombinant factor VIII.

A sixth aspect of the present invention relates to a method of treatingan animal for hemophilia A. This method of treatment includes:administering to an animal exhibiting hemophilia A an effective amountof the recombinant factor VIII of the present invention, whereby theanimal exhibits effective blood clotting following vascular injury.

The present invention demonstrates that a number of residues at theinterface of A1 and C2 domains do not participate in non-covalentbonding, but instead may be destabilizing to factor VIII structure.Replacement of these residues with more hydrophobic residues—with theaim of increasing the buried hydrophobic area and reducing the buriedhydrophilic area—was shown in the accompanying Examples to enhanceinter-domain binding affinity. Stability parameters were assessed byfollowing the activity of the factor VIII variants. Results from thesestudies demonstrated that a number of mutations yielded increasedstability parameters. These stabilized variants of factor VIII andactivated cofactor VIIIa should afford an improved therapeutic fortreatment of hemophilia A.

To explore the role of this region in factor VIII and factor VIIIastability, a variant containing a disulfide bond between A1 and C2domains was generated by mutating Arg121 and Leu2302 to Cys(R121C-L2302C) and a second variant was generated by substituting abulkier hydrophobic group (Ala108Ile) to better occupy a cavity betweenA1 and C2 domains. Disulfide bonding in the R121C-L2302C variantwas >90% efficient as judged by western blots. Binding affinity betweenthe Ala108Ile A1 and A3C1C2 subunits was increased ˜3.7-fold in thevariant compared with WT as judged by changes in fluorescence ofacrylodan labeled-A1 subunits. Factor VIII thermal and chemicalstability were monitored following rates of loss of factor VIII activityat 57° C. or in guanidinium by factor Xa generation assays. The rate ofdecay of factor VIIIa activity was monitored at 23° C. followingactivation by thrombin. Both R121C-L2302C and Ala108Ile variants showedup to ˜4-fold increases in thermal stability but minimal improvements inchemical stability. The purified A1 subunit of Ala1081Ile reconstitutedwith the A3C1C2 subunit showed a ˜4.6-fold increase in thermal stabilitywhile reconstitution of the variant A1 with a truncated A3C1 subunitshowed similar stability values as compared with WT A1. Together, theseresults suggest that altering contacts at this A1-C2 junction bycovalent modification or increasing hydrophobicity increases inter-chainaffinity and functionally enhances factor VIII stability. Moreover, bycombining these mutations with one or more mutations at the A1-A2 and/orA2-A3 domain interfaces, including Asp519Ala, Asp519Val, Glu665Ala,Glu665Val, Glu1984Ala, and Glu1984Val, double and triple mutantsdisplayed ˜2-10 fold increases in the stability of factor VIIIa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows alignment of partial A1 domain sequences of eight mammalianfactor VIII molecules starting from amino acid residue A100 of humanfactor VIII. The partial amino acid sequences are from human factor VIII(residues 100-140 of SEQ ID NO: 2); pig factor VIII (SEQ ID NO: 3);canine factor VIII (SEQ ID NO: 4); mouse factor VIII (SEQ ID NO: 5);rabbit factor VIII (SEQ ID NO: 6); bat factor VIII (SEQ ID NO: 7); ratfactor VIII (SEQ ID NO: 8); and sheep factor VIII (SEQ ID NO: 9). Thefigure also shows the consensus amino acid sequence (SEQ ID NO: 10) forthe partial A1 domain. Underlined residues represent a C2 domaininterface of the A1 domain.

FIG. 2 shows alignment of partial C2 domain sequences from eightmammalian factor VIII molecules starting from amino acid residue Q2231of human factor VIII. The partial amino acid sequences are from humanfactor VIII (residues 2231-2334 of SEQ ID NO: 2); pig factor VIII (SEQID NO: 11); canine factor VIII (SEQ ID NO: 12); mouse factor VIII (SEQID NO: 13); rabbit factor VIII (SEQ ID NO: 14); bat factor VIII (SEQ IDNO: 15); rat factor VIII (SEQ ID NO: 16); and sheep factor VIII (SEQ IDNO: 17). The figure also shows the consensus amino acid sequence (SEQ IDNO: 18) for the partial C2 domain. Underlined residues represent an A1domain interface of the C2 domain.

FIGS. 3A-C illustrate a model of selected residues at the A1-C2 domaininterface. In FIG. 3A, the molecular surface of the factor VIII X-raycrystal structure, drawn by Swiss PDB viewer, shows the A1 domain(residues 1-336) in yellow, A2 domain (residues 373-711) in blue, A3domain (residues 1690-2020) in red, C1 domain (residues 2021-2169) ingreen, and C2 domain (residues 2170-2332) in grey (FIG. 3A). Highermagnification of the A1-C2 contact region (yellow circle) is presentedin FIGS. 3B-C. Side chains of Arg121 and Leu2302 (drawn as stick models)mutated to Cys residues are modeled by Swiss PDB viewer (FIG. 3B). InFIG. 3C, residues surrounding Ala108 (Leu2302, Ala2328, and Gln2329) arehighlighted as stick models. Cβ carbon of Ala108, Cδ of Leu2302, cβ ofAla2328, and Cγ of Gln2329 are indicated by arrows (FIG. 3C, leftpanel). Ala108 mutated to Ile was modeled by Swiss PDB viewer (FIG. 3C,right panel). In the stick models hydrogen, carbon, oxygen, nitrogen,and sulfur are colored as cyan, white, red, blue, and yellow,respectively. Ribbon structures represent α-helix (red) and β-strand(green).

FIG. 4 is a Western blot analysis of R121C-L2302C and WT factor VIII.Purified WT and mutant factor VIII proteins were electrophoresed undernon-reducing (lanes 1, 2, 5, and 6) or reducing (lanes 3, 4, 7, and 8)conditions, transferred, and probed with 58.12 (anti-A1 domain antibody,lane 1-4) or 2D2 (anti-A3 domain antibody, lane 5-8). Protein bands werevisualized by chemifluorescence as described in the accompanyingExamples. Shown are WT (lane 1, 3, 5, and 7) and the R121C-L2302C factorVIII variant (lane 2, 4, 6, and 8) proteins.

FIG. 5 is a graph showing the binding affinity of A1 subunit from WT orAla108Ile for the A3C1C2 subunit. Acrylodan-labeled A1 subunit from WT(circles) or Ala108Ile (triangles) was titrated with A3C1C2 and bindingwas detected by the change of fluorescence as described in theaccompanying Examples. Data points averaged from 3 separatedeterminations were fitted to a quadratic equation curve by non-linearleast squares regression.

FIGS. 6A-C are graphs that illustrate factor VIII R121C-L2302C andAla108Ile variant stability. FIG. 6A shows the factor VIII activitydecay at elevated temperature. Factor VIII (4 nM) was incubated at 57°C. and at the indicated times aliquots were removed and activity wasmeasured by factor Xa generation assays as described in the accompanyingExamples. Data were fitted to a single exponential decay curve bynon-linear least squares regression. FIG. 6B shows the inhibition offactor VIII by guanidinium. FVIII (50 nM) in 0-1.2 M guanidiniumchloride was incubated for 2 hrs at 23° C., diluted 1/50 and factor VIIIactivity was measured by factor Xa generation. Data were fitted to alinear equation by least squares regression. Bars represent standarderror values of 3 measurements. FIG. 6C shows factor VIIIa decay.Thrombin-activated factor VIIIa (1.5 nM) was incubated at 23° C.,aliquots were taken at indicated time points and activity was measuredby factor Xa generation assay as described in the accompanying Examples.Data were fitted to a single exponential decay curve by non-linear leastsquares regression. Symbols denote WT (circles), R121C-L2302C(triangles), and Ala108Ile (squares). Each point represents the valueaveraged from three separate determinations.

FIGS. 7A-B are graphs showing the thermal stability of A1 subunitreconstituted with A3C1C2 or A3C1 subunit. Thermal decay ofreconstituted heterodimer of A1 subunit from WT (circles) or Ala108Ile(triangles) with WT A3C1C2 subunit at 55° C. (FIG. 7A) or A3C1 subunitat 52° C. (FIG. 7B) was detected by residual factor VIIIa activityfollowing addition of A2 subunit as described in the accompanyingExamples. Data were fitted to a single exponential decay curve bynon-linear least squares regression. Data points averaged from 3separate determinations were fitted to a single exponential decay curveto obtain rates.

FIGS. 8A-C are bar graphs comparing the thermal stability (FIG. 8A),chemical stability in the presence of guanidinium (FIG. 8B), and factorVIIIa decay (FIG. 8C) of WT and combination mutants. The combinationmutants include one or more of the A1-C2 domain interface mutations(A108I or R121C-L2302C) in combination with one or more A1-A2 or A2-A3domain interface mutations. Factor VIII activity decay, guanidiniuminhibition, and factor VIIIa decay were measured as described in theaccompanying Examples.

FIG. 9 is a graph comparing the effects of Ala108Ile,Asp519Val/Glu665Val, and the combined Ala108Ile/Asp519Val/Glu665Valvariants, relative to wildtype, as measured using a thrombin generationassay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a recombinant factor VIII having one ormore mutations that result in enhanced stability of factor VIII, inparticular improved thermal and/or chemical stability of factor VIII.

The recombinant factor VIII of the present invention can be prepared bymodifying the amino acid sequence of a wild-type factor VIII or a mutantfactor VIII that has otherwise been modified to affect other propertiesof the factor VIII, such as antigenicity, factor VIIIa stability,circulating half-life, protein secretion, affinity for factor IXa and/orfactor X, altered factor VIII-inactivation cleavage sites,immunogenicity, shelf-life, etc.

Suitable wild-type factor VIII that can be modified in accordance withthe present invention can be from various animals including, withoutlimitation, mammals such as humans (see, e.g., GenBank Accession Nos.AAA52484 (amino acid) and K01740 (nucleotide); and GenBank AccessionNos. CAD97566 (amino acid) and AX746360 (nucleotide), which are herebyincorporated by reference in their entirety), rats (see, e.g., GenBankAccession Nos. AAQ21580 (amino acid) and AY362193 (nucleotide), whichare hereby incorporated by reference in their entirety), mice (see,e.g., GenBank Accession Nos. AAA37385 (amino acid) and L05573(nucleotide), which are hereby incorporated by reference in theirentirety), guinea pigs, dogs (see, e.g., GenBank Accession Nos. AAB87412(amino acid) and AF016234 (nucleotide); and GenBank Accession Nos.AAC05384 (amino acid) and AF049489 (nucleotide), which are herebyincorporated by reference in their entirety), cats, monkeys, chimpanzees(see, e.g., GenBank Accession Nos. XP_(—)529212 (amino acid) andXM_(—)529212 (nucleotide), which are hereby incorporated by reference intheir entirety), orangutans, cows, horses, sheep, pigs (see, e.g.,GenBank Accession Nos. NP_(—)999332 (amino acid) and NM_(—)214167(nucleotide), which are hereby incorporated by reference in theirentirety), goats, rabbits, and chickens. These and other sequences arealso available electronically via the Haemophilia A Mutation, Structure,Test and Resource Site (or HAMSTeRS), which further provides analignment of human, porcine, murine, and canine factor VIII proteins.Thus, the conservation and homology among mammalian factor VIII proteinsis well known.

By way of example, the human factor VIII cDNA nucleotide and predictedamino acid sequences are shown below in SEQ ID NOs: 1 and 2,respectively. Human factor VIII is synthesized as an approximately 300kDa single chain protein with internal sequence homology that definesthe “domain” sequence NH₂-A1-A2-B-A3-C1-C2-COOH. In a factor VIIImolecule, a “domain,” as used herein, is a continuous sequence of aminoacids that is defined by internal amino acid sequence identity and sitesof proteolytic cleavage by thrombin. Unless otherwise specified, factorVIII domains include the following amino acid residues, when thesequences are aligned with the human amino acid sequence (SEQ ID NO: 2):

-   -   A1, residues Ala₁-Arg₃₇₂;    -   A2, residues Ser₃₇₃-Arg₇₄₀;    -   B, residues Ser₇₄₁-Arg₁₆₄₈;    -   A3, residues Ser₁₆₉₀-Ile₂₀₃₂;    -   C1, residues Arg₂₀₃₃-Asn₂₁₇₂; and    -   C2, residues Ser₂₁₇₃-Tyr₂₃₃₂.

The A3-C1-C2 sequence includes residues Ser₁₆₉₀-Tyr₂₃₃₂. The remainingsequence, residues Glu₁₆₄₉-Arg₁₆₈₉, is usually referred to as the factorVIII light chain activation peptide. Factor VIII is proteolyticallyactivated by thrombin or factor Xa, which dissociates it from vonWillebrand factor, forming factor VIIIa, which has procoagulantfunction. The biological function of factor VIIIa is to increase thecatalytic efficiency of factor IXa toward factor X activation by severalorders of magnitude. Thrombin-activated factor VIIIa is a 160 kDaA1/A2/A3-C1-C2 heterotrimer that forms a complex with factor IXa andfactor X on the surface of platelets or monocytes. A “partial domain” asused herein is a continuous sequence of amino acids forming part of adomain.

As used herein, a residue corresponding to a particular position refersto the residue at that position in wild type human factor VIII; howeverthe same position will retain its numbering even though the residue maybe at a different location in a recombinant factor VIII of the presentinvention. For example, a B domainless factor VIII lacks the B domainyet it still retains the conventional residue numbering for domains A3,C1, and C2.

The cDNA encoding wild-type human factor VIII has a nucleotide sequenceof SEQ ID NO: 1, as follows:

   1 GCCACCAGAA GATACTACCT GGGTGCAGTG GAACTGTCAT GGGACTATAT  51 GCAAAGTGAT CTCGGTGAGC TGCCTGTGGA CGCAAGATTT CCTCCTAGAG 101 TGCCAAAATC TTTTCCATTC AACACCTCAG TCGTGTACAA AAAGACTCTG 151 TTTGTAGAAT TCACGGATCA CCTTTTCAAC ATCGCTAAGC CAAGGCCACC 201 CTGGATGGGT CTGCTAGGTC CTACCATCCA GGCTGAGGTT TATGATACAG 251 TGGTCATTAC ACTTAAGAAC ATGGCTTCCC ATCCTGTCAG TCTTCATGCT 301 GTTGGTGTAT CCTACTGGAA AGCTTCTGAG GGAGCTGAAT ATGATGATCA 351 GACCAGTCAA AGGGAGAAAG AAGATGATAA AGTCTTCCCT GGTGGAAGCC 401 ATACATATGT CTGGCAGGTC CTGAAAGAGA ATGGTCCAAT GGCCTCTGAC 451 CCACTGTGCC TTACCTACTC ATATCTTTCT CATGTGGACC TGGTAAAAGA 501 CTTGAATTCA GGCCTCATTG GAGCCCTACT AGTATGTAGA GAAGGGAGTC 551 TGGCCAAGGA AAAGACACAG ACCTTGCACA AATTTATACT ACTTTTTGCT 601 GTATTTGATG AAGGGAAAAG TTGGCACTCA GAAACAAAGA ACTCCTTGAT 651 GCAGGATAGG GATGCTGCAT CTGCTCGGGC CTGGCCTAAA ATGCACACAG 701 TCAATGGTTA TGTAAACAGG TCTCTGCCAG GTCTGATTGG ATGCCACAGG 751 AAATCAGTCT ATTGGCATGT GATTGGAATG GGCACCACTC CTGAAGTGCA 801 CTCAATATTC CTCGAAGGTC ACACATTTCT TGTGAGGAAC CATCGCCAGG 851 CGTCCTTGGA AATCTCGCCA ATAACTTTCC TTACTGCTCA AACACTCTTG 901 ATGGACCTTG GACAGTTTCT ACTGTTTTGT CATATCTCTT CCCACCAACA 951 TGATGGCATG GAAGCTTATG TCAAAGTAGA CAGCTGTCCA GAGGAACCCC1001 AACTACGAAT GAAAAATAAT GAAGAAGCGG AAGACTATGA TGATGATCTT1051 ACTGATTCTG AAATGGATGT GGTCAGGTTT GATGATGACA ACTCTCCTTC1101 CTTTATCCAA ATTCGCTCAG TTGCCAAGAA GCATCCTAAA ACTTGGGTAC1151 ATTACATTGC TGCTGAAGAG GAGGACTGGG ACTATGCTCC CTTAGTCCTC1201 GCCCCCGATG ACAGAAGTTA TAAAAGTCAA TATTTGAACA ATGGCCCTCA1251 GCGGATTGGT AGGAAGTACA AAAAAGTCCG ATTTATGGCA TACACAGATG1301 AAACCTTTAA GACTCGTGAA GCTATTCAGC ATGAATCAGG AATCTTGGGA1351 CCTTTACTTT ATGGGGAAGT TGGAGACACA CTGTTGATTA TATTTAAGAA1401 TCAAGCAAGC AGACCATATA ACATCTACCC TCACGGAATC ACTGATGTCC1451 GTCCTTTGTA TTCAAGGAGA TTACCAAAAG GTGTAAAACA TTTGAAGGAT1501 TTTCCAATTC TGCCAGGAGA AATATTCAAA TATAAATGGA CAGTGACTGT1551 AGAAGATGGG CCAACTAAAT CAGATCCTCG GTGCCTGACC CGCTATTACT1601 CTAGTTTCGT TAATATGGAG AGAGATCTAG CTTCAGGACT CATTGGCCCT1651 CTCCTCATCT GCTACAAAGA ATCTGTAGAT CAAAGAGGAA ACCAGATAAT1701 GTCAGACAAG AGGAATGTCA TCCTGTTTTC TGTATTTGAT GAGAACCGAA1751 GCTGGTACCT CACAGAGAAT ATACAACGCT TTCTCCCCAA TCCAGCTGGA1801 GTGCAGCTTG AGGATCCAGA GTTCCAAGCC TCCAACATCA TGCACAGCAT1851 CAATGGCTAT GTTTTTGATA GTTTGCAGTT GTCAGTTTGT TTGCATGAGG1901 TGGCATACTG GTACATTCTA AGCATTGGAG CACAGACTGA CTTCCTTTCT1951 GTCTTCTTCT CTGGATATAC CTTCAAACAC AAAATGGTCT ATGAAGACAC2001 ACTCACCCTA TTCCCATTCT CAGGAGAAAC TGTCTTCATG TCGATGGAAA2051 ACCCAGGTCT ATGGATTCTG GGGTGCCACA ACTCAGACTT TCGGAACAGA2101 GGCATGACCG CCTTACTGAA GGTTTCTAGT TGTGACAAGA ACACTGGTGA2151 TTATTACGAG GACAGTTATG AAGATATTTC AGCATACTTG CTGAGTAAAA2201 ACAATGCCAT TGAACCAAGA AGCTTCTCCC AGAATTCAAG ACACCCTAGC2251 ACTAGGCAAA AGCAATTTAA TGCCACCACA ATTCCAGAAA ATGACATAGA2301 GAAGACTGAC CCTTGGTTTG CACACAGAAC ACCTATGCCT AAAATACAAA2351 ATGTCTCCTC TAGTGATTTG TTGATGCTCT TGCGACAGAG TCCTACTCCA2401 CATGGGCTAT CCTTATCTGA TCTCCAAGAA GCCAAATATG AGACTTTTTC2451 TGATGATCCA TCACCTGGAG CAATAGACAG TAATAACAGC CTGTCTGAAA2501 TGACACACTT CAGGCCACAG CTCCATCACA GTGGGGACAT GGTATTTACC2551 CCTGAGTCAG GCCTCCAATT AAGATTAAAT GAGAAACTGG GGACAACTGC2601 AGCAACAGAG TTGAAGAAAC TTGATTTCAA AGTTTCTAGT ACATCAAATA2651 ATCTGATTTC AACAATTCCA TCAGACAATT TGGCAGCAGG TACTGATAAT2701 ACAAGTTCCT TAGGACCCCC AAGTATGCCA GTTCATTATG ATAGTCAATT2751 AGATACCACT CTATTTGGCA AAAAGTCATC TCCCCTTACT GAGTCTGGTG2801 GACCTCTGAG CTTGAGTGAA GAAAATAATG ATTCAAAGTT GTTAGAATCA2851 GGTTTAATGA ATAGCCAAGA AAGTTCATGG GGAAAAAATG TATCGTCAAC2901 AGAGAGTGGT AGGTTATTTA AAGGGAAAAG AGCTCATGGA CCTGCTTTGT2951 TGACTAAAGA TAATGCCTTA TTCAAAGTTA GCATCTCTTT GTTAAAGACA3001 AACAAAACTT CCAATAATTC AGCAACTAAT AGAAAGACTC ACATTGATGG3051 CCCATCATTA TTAATTGAGA ATAGTCCATC AGTCTGGCAA AATATATTAG3101 AAAGTGACAC TGAGTTTAAA AAAGTGACAC CTTTGATTCA TGACAGAATG3151 CTTATGGACA AAAATGCTAC AGCTTTGAGG CTAAATCATA TGTCAAATAA3201 AACTACTTCA TCAAAAAACA TGGAAATGGT CCAACAGAAA AAAGAGGGCC3251 CCATTCCACC AGATGCACAA AATCCAGATA TGTCGTTCTT TAAGATGCTA3301 TTCTTGCCAG AATCAGCAAG GTGGATACAA AGGACTCATG GAAAGAACTC3351 TCTGAACTCT GGGCAAGGCC CCAGTCCAAA GCAATTAGTA TCCTTAGGAC3401 CAGAAAAATC TGTGGAAGGT CAGAATTTCT TGTCTGAGAA AAACAAAGTG3451 GTAGTAGGAA AGGGTGAATT TACAAAGGAC GTAGGACTCA AAGAGATGGT3501 TTTTCCAAGC AGCAGAAACC TATTTCTTAC TAACTTGGAT AATTTACATG3551 AAAATAATAC ACACAATCAA GAAAAAAAAA TTCAGGAAGA AATAGAAAAG3601 AAGGAAACAT TAATCCAAGA GAATGTAGTT TTGCCTCAGA TACATACAGT3651 GACTGGCACT AAGAATTTCA TGAAGAACCT TTTCTTACTG AGCACTAGGC3701 AAAATGTAGA AGGTTCATAT GACGGGGCAT ATGCTCCAGT ACTTCAAGAT3751 TTTAGGTCAT TAAATGATTC AACAAATAGA ACAAAGAAAC ACACAGCTCA3801 TTTCTCAAAA AAAGGGGAGG AAGAAAACTT GGAAGGCTTG GGAAATCAAA3851 CCAAGCAAAT TGTAGAGAAA TATGCATGCA CCACAAGGAT ATCTCCTAAT3901 ACAAGCCAGC AGAATTTTGT CACGCAACGT AGTAAGAGAG CTTTGAAACA3951 ATTCAGACTC CCACTAGAAG AAACAGAACT TGAAAAAAGG ATAATTGTGG4001 ATGACACCTC AACCCAGTGG TCCAAAAACA TGAAACATTT GACCCCGAGC4051 ACCCTCACAC AGATAGACTA CAATGAGAAG GAGAAAGGGG CCATTACTCA4101 GTCTCCCTTA TCAGATTGCC TTACGAGGAG TCATAGCATC CCTCAAGCAA4151 ATAGATCTCC ATTACCCATT GCAAAGGTAT CATCATTTCC ATCTATTAGA4201 CCTATATATC TGACCAGGGT CCTATTCCAA GACAACTCTT CTCATCTTCC4251 AGCAGCATCT TATAGAAAGA AAGATTCTGG GGTCCAAGAA AGCAGTCATT4301 TCTTACAAGG AGCCAAAAAA AATAACCTTT CTTTAGCCAT TCTAACCTTG4351 GAGATGACTG GTGATCAAAG AGAGGTTGGC TCCCTGGGGA CAAGTGCCAC4401 AAATTCAGTC ACATACAAGA AAGTTGAGAA CACTGTTCTC CCGAAACCAG4451 ACTTGCCCAA AACATCTGGC AAAGTTGAAT TGCTTCCAAA AGTTCACATT4501 TATCAGAAGG ACCTATTCCC TACGGAAACT AGCAATGGGT CTCCTGGCCA4551 TCTGGATCTC GTGGAAGGGA GCCTTCTTCA GGGAACAGAG GGAGCGATTA4601 AGTGGAATGA AGCAAACAGA CCTGGAAAAG TTCCCTTTCT GAGAGTAGCA4651 ACAGAAAGCT CTGCAAAGAC TCCCTCCAAG CTATTGGATC CTCTTGCTTG4701 GGATAACCAC TATGGTACTC AGATACCAAA AGAAGAGTGG AAATCCCAAG4751 AGAAGTCACC AGAAAAAACA GCTTTTAAGA AAAAGGATAC CATTTTGTCC4801 CTGAACGCTT GTGAAAGCAA TCATGCAATA GCAGCAATAA ATGAGGGACA4851 AAATAAGCCC GAAATAGAAG TCACCTGGGC AAAGCAAGGT AGGACTGAAA4901 GGCTGTGCTC TCAAAACCCA CCAGTCTTGA AACGCCATCA ACGGGAAATA4951 ACTCGTACTA CTCTTCAGTC AGATCAAGAG GAAATTGACT ATGATGATAC5001 CATATCAGTT GAAATGAAGA AGGAAGATTT TGACATTTAT GATGAGGATG5051 AAAATCAGAG CCCCCGCAGC TTTCAAAAGA AAACACGACA CTATTTTATT5101 GCTGCAGTGG AGAGGCTCTG GGATTATGGG ATGAGTAGCT CCCCACATGT5151 TCTAAGAAAC AGGGCTCAGA GTGGCAGTGT CCCTCAGTTC AAGAAAGTTG5201 TTTTCCAGGA ATTTACTGAT GGCTCCTTTA CTCAGCCCTT ATACCGTGGA5251 GAACTAAATG AACATTTGGG ACTCCTGGGG CCATATATAA GAGCAGAAGT5301 TGAAGATAAT ATCATGGTAA CTTTCAGAAA TCAGGCCTCT CGTCCCTATT5351 CCTTCTATTC TAGCCTTATT TCTTATGAGG AAGATCAGAG GCAAGGAGCA5401 GAACCTAGAA AAAACTTTGT CAAGCCTAAT GAAACCAAAA CTTACTTTTG5451 GAAAGTGCAA CATCATATGG CACCCACTAA AGATGAGTTT GACTGCAAAG5501 CCTGGGCTTA TTTCTCTGAT GTTGACCTGG AAAAAGATGT GCACTCAGGC5551 CTGATTGGAC CCCTTCTGGT CTGCCACACT AACACACTGA ACCCTGCTCA5601 TGGGAGACAA GTGACAGTAC AGGAATTTGC TCTGTTTTTC ACCATCTTTG5651 ATGAGACCAA AAGCTGGTAC TTCACTGAAA ATATGGAAAG AAACTGCAGG5701 GCTCCCTGCA ATATCCAGAT GGAAGATCCC ACTTTTAAAG AGAATTATCG5751 CTTCCATGCA ATCAATGGCT ACATAATGGA TACACTACCT GGCTTAGTAA5801 TGGCTCAGGA TCAAAGGATT CGATGGTATC TGCTCAGCAT GGGCAGCAAT5851 GAAAACATCC ATTCTATTCA TTTCAGTGGA CATGTGTTCA CTGTACGAAA5901 AAAAGAGGAG TATAAAATGG CACTGTACAA TCTCTATCCA GGTGTTTTTG5951 AGACAGTGGA AATGTTACCA TCCAAAGCTG GAATTTGGCG GGTGGAATGC6001 CTTATTGGCG AGCATCTACA TGCTGGGATG AGCACACTTT TTCTGGTGTA6051 CAGCAATAAG TGTCAGACTC CCCTGGGAAT GGCTTCTGGA CACATTAGAG6101 ATTTTCAGAT TACAGCTTCA GGACAATATG GACAGTGGGC CCCAAAGCTG6151 GCCAGACTTC ATTATTCCGG ATCAATCAAT GCCTGGAGCA CCAAGGAGCC6201 CTTTTCTTGG ATCAAGGTGG ATCTGTTGGC ACCAATGATT ATTCACGGCA6251 TCAAGACCCA GGGTGCCCGT CAGAAGTTCT CCAGCCTCTA CATCTCTCAG6301 TTTATCATCA TGTATAGTCT TGATGGGAAG AAGTGGCAGA CTTATCGAGG6351 AAATTCCACT GGAACCTTAA TGGTCTTCTT TGGCAATGTG GATTCATCTG6401 GGATAAAACA CAATATTTTT AACCCTCCAA TTATTGCTCG ATACATCCGT6451 TTGCACCCAA CTCATTATAG CATTCGCAGC ACTCTTCGCA TGGAGTTGAT6501 GGGCTGTGAT TTAAATAGTT GCAGCATGCC ATTGGGAATG GAGAGTAAAG6551 CAATATCAGA TGCACAGATT ACTGCTTCAT CCTACTTTAC CAATATGTTT6601 GCCACCTGGT CTCCTTCAAA AGCTCGACTT CACCTCCAAG GGAGGAGTAA6651 TGCCTGGAGA CCTCAGGTGA ATAATCCAAA AGAGTGGCTG CAAGTGGACT6701 TCCAGAAGAC AATGAAAGTC ACAGGAGTAA CTACTCAGGG AGTAAAATCT6751 CTGCTTACCA GCATGTATGT GAAGGAGTTC CTCATCTCCA GCAGTCAAGA6801 TGGCCATCAG TGGACTCTCT TTTTTCAGAA TGGCAAAGTA AAGGTTTTTC6851 AGGGAAATCA AGACTCCTTC ACACCTGTGG TGAACTCTCT AGACCCACCG6901 TTACTGACTC GCTACCTTCG AATTCACCCC CAGAGTTGGG TGCACCAGAT6951 TGCCCTGAGG ATGGAGGTTC TGGGCTGCGA GGCACAGGAC CTCTACTGA

The wild-type human factor VIII encoded by SEQ ID NO: 1 has an aminoacid sequence of SEQ ID NO: 2, as follows:

   1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL  51 FVEFTVHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA 101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD 151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA 201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR 251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL 301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL 351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL 401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG 451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD 501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP 551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG 601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS 651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR 701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNSRHPS 751 TRQKQFNATT IPENDIEKTD PWFAHRTPMP KIQNVSSSDL LMLLRQSPTP 801 HGLSLSDLQE AKYETFSDDP SPGAIDSNNS LSEMTHFRPQ LHHSGDMVFT 851 PESGLQLRLN EKLGTTAATE LKKLDFKVSS TSNNLISTIP SDNLAAGTDN 901 TSSLGPPSMP VHYDSQLDTT LFGKKSSPLT ESGGPLSLSE ENNDSKLLES 951 GLMNSQESSW GKNVSSTESG RLFKGKRAHG PALLTKDNAL FKVSISLLKT1001 NKTSNNSATN RKTHIDGPSL LIENSPSVWQ NILESDTEFK KVTPLIHDRM1051 LMDKNATALR LNHMSNKTTS SKNMEMVQQK KEGPIPPDAQ NPDMSFFKML1101 FLPESARWIQ RTHGKNSLNS GQGPSPKQLV SLGPEKSVEG QNFLSEKNKV1151 VVGKGEFTKD VGLKEMVFPS SRNLFLTNLD NLHENNTHNQ EKKIQEEIEK1201 KETLIQENVV LPQIHTVTGT KNFMKNLFLL STRQNVEGSY EGAYAPVLQD1251 FRSLNDSTNR TKKHTAHFSK KGEEENLEGL GNQTKQIVEK YACTTRISPN1301 TSQQNFVTQR SKRALKQFRL PLEETELEKR IIVDDTSTQW SKNMKHLTPS1351 TLTQIDYNEK EKGAITQSPL SDCLTRSHSI PQANRSPLPI AKVSSFPSIR1401 PIYLTRVLFQ DNSSHLPAAS YRKKDSGVQE SSHFLQGAKK NNLSLAILTL1451 EMTGDQREVG SLGTSATNSV TYKKVENTVL PKPDLPKTSG KVELLPKVHI1501 YQKDLFPTET SNGSPGHLDL VEGSLLQGTE GAIKWNEANR PGKVPFLRVA1551 TESSAKTPSK LLDPLAWDNH YGTQIPKEEW KSQEKSPEKT AFKKKDTILS1601 LNACESNHAI AAINEGQNKP EIEVTWAKQG RTERLCSQNP PVLKRHQREI1651 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI1701 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG1751 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA1801 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG1851 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR1901 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN1951 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC2001 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL2051 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ2101 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR2151 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF2201 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS2251 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP2301 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY

A first aspect of the present invention relates to a recombinant factorVIII that includes one or more mutations at an interface of A1 and C2domains of the recombinant factor VIII. This mutation results inenhanced stability, particularly enhanced thermal and/or chemicalstability, of factor VIII. The one or more mutations includesubstitution of one or more amino acid residues with an amino acidresidue having a higher hydrophobicity, or substitution of two or moreamino acid residues with Cysteine to afford a disulfide bond between theA1 and C2 domains.

As used herein, an amino acid having a higher hydrophobicity refers to aresidue having a higher measurement or ranking of hydrophobicityrelative to a particular wild type residue at the location of interest.The hydrophobic effect represents the tendency of water to excludenon-polar molecules. Hydropathy scale is a ranking list for the relativehydrophobicity of amino acid residues and proteins. The “hydropathyindex” of a protein or amino acid is a number representing itshydrophilic or hydrophobic properties. Different methods have been usedin the art to calculate the relative hydrophobicity of amino acidresidues and proteins (Kyte et al., “A Simple Method for Displaying theHydropathic Character of a Protein,” J. Mol. Biol. 157: 105-32 (1982);Eisenberg D, “Three-dimensional Structure of Membrane and SurfaceProteins,” Ann. Rev. Biochem. 53: 595-623 (1984); Rose et al., “HydrogenBonding, Hydrophobicity, Packing, and Protein Folding,” Annu. Rev.Biomol. Struct. 22: 381-415 (1993); Kauzmann, “Some Factors in theInterpretation of Protein Denaturation,” Adv. Protein Chem. 14: 1-63(1959), which are hereby incorporated by reference in their entirety).Any one of these hydrophobicity scales can be used for the purposes ofthe present invention; however, the Kyte-Doolittle hydrophobicity scaleis perhaps the most often referenced scale. The hydropathy index isdirectly proportional to the hydrophobicity of the amino acid or theprotein.

As used herein, the term “interface” is used to describe a proteinsurface where the atoms of the protein come in contact with the solvent(solvent-protein interface) or with another domain (domain interface).Domain interfaces can be either inter-domain (between domains) orintra-domain (within domains). Various methods are known in the art toidentify interfaces. For example, the geometric distance between atomsthat belong to same or different domains can be used to identifyintra-domain or inter-domain interfaces (structural information, such asatomic coordinates, is available at the Protein Databank; Berman et al.,“The Protein Data Bank,” Nucleic Acid Res 28:235-242 (2000), which ishereby incorporated by reference in its entirety). Another approach isthe Accessible Surface Area (ASA), which detects the buried region of aprotein that is detached from a solvent (Jones et al., “Analysis ofProtein-protein Interaction Sites Using Surface Patches,” J Mol Biol272:121-132 (1997), which is hereby incorporated by reference in itsentirety). A further approach is the Voronoi diagram, a computationalgeometry method that uses a mathematical definition of interface regions(Ban et al., “Interface Surfaces for Protein-protein Complexes,”Proceedings of the Research in Computational Molecular Biology, SanDiego 27-31 (2004); Poupon A, “Voronoi and Voronoi-Related Tessellationsin Studies of Protein Structure and Interaction,” Curr Opin Struct Biol14:233-241 (2004); Kim et al., “Euclidean Voronoi Diagrams of 3D Spheresand Applications to Protein Structure Analysis,” Japan Journal ofIndustrial and Applied Mathematics 2005, 22:251-265 (2005), which arehereby incorporated by reference in their entirety). As describedherein, the factor VIII crystal structure (Shen et al., “The TertiaryStructure and Domain Organization of Coagulation Factor VIII,” Blood111:1240-1247 (2008); Ngo et al., “Crystal Structure of Human FactorVIII: Implications for the Formation of the Factor IXa:Factor VIIIaComplex,” Structure 16:597-606 (2008), which is hereby incorporated byreference in its entirety) can be modeled using Swiss PDB Viewer toidentify residues that upon substitution with Cysteine will allow fordisulfide bond formation (see FIG. 3B) or residues that are suitable forsubstitution with an amino acid having higher hydrophobicity (see FIG.3C).

As used herein, a region of the A1 domain that interfaces with the C2domain is referred to as a “C2 domain interface” and a region of the C2domain that interfaces with the A1 domain is referred to as an “A1domain interface.”

The wild type (WT) factor VIII can be such that the A1 domain of the WTfactor VIII includes a C2 domain interface having the amino acidsequence of (i) KXS (aa 8-10 of SEQ ID NO: 10) where “X” is A or S; (ii)SXXE (aa 20-23 of SEQ ID NO: 10) where “X” at the 2^(nd) position can beQ, K, or P, and “X” at the 3^(rd) position can be R, K, M, T, or A;and/or (iii) TYXW (aa 36-39 of SEQ ID NO: 10), where “X” at the 3^(rd)position can be V or A. These are illustrated in FIG. 1.

The WT factor VIII can be such that the C2 domain of the WT factor VIIIincludes an A1 domain interface having the amino acid sequence of (i)XVT (aa 9-11 of SEQ ID NO: 18), where “X” is K or R; (ii) PPXX (aa 68-71of SEQ ID NO: 18), where “X” at the 3^(rd) position is L or R, and “X”at the 4^(th) position is L, F, or V; and/or (iii) XXQX (aa 96-99 of SEQID NO: 18), where the “X” at the 1^(st) position is E or D, “X” at the2^(nd) position is A or T, and “X” at the 4^(th) position is D or Q.These are illustrated in FIG. 2.

According to one embodiment, one or both of the A1 domain interface andC2 domain interface include a substitution to introduce a Cysteineresidue. In preferred embodiments, the substitution of amino acidresidues at the interface of A1 and C2 domains is carried out such thatat least a pair of amino acids are substituted with Cysteine. In thisembodiment, the pair of Cysteine residues form an inter-domain (A1 to C2domain) disulfide bond such that the stability of the recombinant factorVIII is enhanced.

In another embodiment of the recombinant factor VIII of the presentinvention, the one or more mutations at the interface of A1 and C2domains of the recombinant factor VIII is the replacement of one or moreamino acid residues with an amino acid incapable of forming disulfidebonds but having a higher hydrophobicity index.

In a further embodiment, multiple mutations are introduced at severalinterfaces of the A1 and C2 domains of factor VIII, including: (i) apair of substitutions to introduce a pair of Cysteine residues that arecapable of forming an inter-domain (A1 to C2 domain) disulfide bond; and(ii) the replacement of one or more amino acid residues with an aminoacid incapable of forming disulfide bonds but having a higherhydrophobicity index.

The recombinant factor VIII according to several embodiments of thepresent invention are characterized by an A1 domain that includes a C2domain interface having the amino acid sequence of (i) KXS (SEQ ID NO:19), where “X” is T, G, A, M, C, F, L, V, or I; (ii) SXXX (SEQ ID NO:20), where “X” at the 2^(nd) position is wild type (Q, K, or P) or E, D,N, H, Y, W, S, T, G, A, M, C, F, L, V, or I, “X” at the 3^(rd) positioncan be any amino acid other than R or preferably any amino acid otherthan R, K, M, T, or A; and “X” at the 4^(th) position is wild type (E)or Q, D, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I; and/or (iii)TYXW (SEQ ID NO: 21), where “X” is M, C, F, L, V, or I. In at least oneof the C2 domain interfaces, one of the X residues represents asubstitution of a wild type residue.

The recombinant factor VIII according to several embodiments of thepresent invention are characterized by a C2 domain that includes an A1domain interface having the amino acid sequence of (i) XVT (SEQ ID NO:22), where “X” can be any amino acid other than K or R; (ii) PPXX (SEQID NO: 23), where “X” at the 3^(rd) position can be any amino acidbesides L or R and “X” at the 4^(th) position is L, V, or I; and/or(iii) XXQX (SEQ ID NO: 24), where “X” at the 1^(st) position is wildtype (E or D) or Q, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I, “X”at the 2^(nd) position is wild type (A or T) or G, M, C, F, L, V, or I,and “X” at the 4^(th) position is wild type (D or Q) or N, H, P, Y, W,S, T, G, A, M, C, F, L, V, or I. In at least one of the A1 domaininterfaces, one of the X residues represents a substitution of a wildtype residue.

In certain embodiments, where a disulfide linkage is formed between A1and C2 domains using a cysteine substitution, the cysteine substitutionoccurs at residue 121 of human factor VIII (i.e., the C2 domaininterface is SEQ ID NO: 20, where X at the third position is cysteine)and residue 2302 of human factor VIII (i.e., the A1 domain interface isSEQ ID NO: 23, where X at the fourth position is cysteine). In otherembodiments, the cysteine substitution occurs at a residue other thanresidues 121 and 2302 of human factor VIII.

One embodiment of the recombinant factor VIII of the present inventionincludes an A1 domain having a C2 domain interface that includes theamino acid sequence KXS (SEQ ID NO: 19), where the second residue(corresponding to position 108 of SEQ ID NO: 2) is Valine, Isoleucine,or Leucine.

A further embodiment of the recombinant factor VIII of the presentinvention includes a C2 domain having an A1 domain interface thatincludes the amino acid sequence of XVT (SEQ ID NO: 22), where X(corresponding to position 2328 of SEQ ID NO: 2) is other than Lysine orArginine.

Another embodiment of the recombinant factor VIII includes an A1 domainhaving a C2 domain interface that includes the amino acid sequence ofSXXE (SEQ ID NO: 25), where the second residue can be Q, K, P, E, D, N,H, Y, W, S, T, G, A, M, C, F, L, V, or I and the third residue(corresponding to position 121 of SEQ ID NO: 2) is cysteine; and a C2domain having an A1 domain interface that includes the amino acidsequence of PPXX (SEQ ID NO: 23), where X at the 3^(rd) position is anyamino acid besides L or R, and X at the 4^(th) position (correspondingto position 2302 of SEQ ID NO: 2) is cysteine.

Yet another embodiment of the recombinant factor includes a C2 domainhaving an A1 domain interface that includes the amino acid sequence ofXXQX (SEQ ID NO: 24), where the 1^(st) residue is E, D, Q, N, H, P, Y,W, S, T, G, A, M, C, F, L, V, or I; the 2^(nd) residue (corresponding toposition 2328 of SEQ ID NO: 2) is Valine, Isoleucine, or Leucine; andthe 4^(th) residue is D, Q, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, orI.

The recombinant factor VIII according to the present invention can alsohave more than one mutation as described supra. In one preferredembodiment the recombinant factor VIII has two or more amino acidsubstitutions.

According to one embodiment, the recombinant factor VIII includes (i) anA1 domain having a C2 domain interface that includes the amino acidsequence of KXS (SEQ ID NO: 19), where X (corresponding to position 108of SEQ ID NO: 2) is Isoleucine, Leucine, or Valine; and (ii) a C2 domainhaving an A1 domain interface that includes the amino acid sequence XXQX(SEQ ID NO: 24), where the 1^(st) and 4^(th) residues are as describedabove and the 2^(nd) residue (corresponding to position 2328 of SEQ IDNO: 2) is Isoleucine, Leucine, or Valine.

Suitable mutant factor VIII sequences that can be modified in accordancewith the present invention can also include any previously known orsubsequently identified mutant factor VIII sequences that have modifiedproperties with regard to various attributes, including, withoutlimitation, antigenicity, circulating half-life, factor VIIIa stability,protein secretion, affinity for factor IXa and/or factor X, alteredfactor VIII-inactivation cleavage sites, altered activated Protein Ccleavage sites, enhanced specific activity of factor VIIIa,immunogenicity, and shelf-life.

In one embodiment the recombinant factor VIII of the present inventionfurther comprises one or more of (i) factor IXa and/or factor X bindingdomains modified to enhance the affinity of the recombinant factor VIIIfor one or both of factor IXa and factor X; (ii) modified sites thatenhance secretion in culture; (iii) modified serum protein binding sitesthat enhance the circulating half-life thereof; (iv) at least oneglycosylation recognition sequence that is effective in decreasingantigenicity and/or immunogenicity thereof; (v) a modified A1 domaincalcium-binding site that improves specific activity of the recombinantfactor VIIIa; (vi) modified activated protein C-cleavage site; (vii) amodified A1 and A2 domain interface to enhance factor VIIIa stability;and (viii) a modified A2 and A3 domain interface to enhance factor VIIIastability.

The recombinant factor VIII of the present invention can be one that hasa combination of mutations, including one or more mutations at aninterface of A1 and C2 domains of recombinant factor VIII as describedsupra and one or more mutations at the A1 and A2 domain interface and/orthe A2 and A3 domain interface. Such a recombinant factor VIII inaddition to the one or more mutations at the A1 and C2 domain interfacealso includes substitution of one or more charged amino acid residueswith a hydrophobic amino acid residue at either or both of the A1 and A2or A2 and A3 domain interfaces.

Preferably, the charged residue to be replaced is either a Glu or Aspresidue that does not participate in hydrogen bonding between the A1 andA2 or A2 and A3 domains. The hydrophobic amino acid residue thatreplaces the charged residue can be any of Ala, Val, Ile, Leu, Met, Phe,or Trp. Particularly preferred recombinant factor VIII of the presentinvention includes a substitution of the residue corresponding to Glu287of wild type factor VIII, a substitution of the residue corresponding toAsp302 of wild type factor VIII, a substitution of the residuecorresponding to Asp519 of wild type factor VIII, a substitution of theresidue corresponding to Glu665 of wild type factor VIII, a substitutionof the residue corresponding to Glu1984 of wild type factor VIII, orcombinations thereof. The D302A, E287A, E665A, E665V, D519A, D519V,E1984A, and E1984V substitutions are preferred for achieving arecombinant factor VIII that has enhanced stability of both factor VIIIand factor VIIIa. Preferred combinations of these substitutions include,without limitation, those corresponding to D519AE665V, D519VE665V, andD519VE1984A mutants, as well as D519AE665VE1984A and D519VE665VE1984Amutants. The enhanced stability of these mutants is believed to beachieved by stabilizing the inter-domain interface in factor VIII aswell as reducing A2 subunit dissociation from A1/A3C1C2 as compared towild type factor VIIIa. Exemplary mutants of this type are described inU.S. Patent Application Publ. No. US2009/0118184 to Fay et al., which ishereby incorporated by reference in its entirety.

Examples of mutant factor VIII possessing substitutions at the A1-C2domain interface as well as one or both of the A1-A2 and A2-A3 domaininterfaces include, without limitation, A108ID519AE665V,A108ID519VE665V, A108ID519VE1984A, A108ID519AE665VE1984A,A108ID519VE665VE1984A, R121C-L2302C/D519AE665V, R121C-L2302C/D519VE665V,R121C-L2302C/D519VE1984A, R121C-L2302C/D519AE665VE1984A, andR121C-L2302C/D519VE665VE1984A. Each of these mutant factor VIII can beexpressed in a B-domainless form, as described below.

Another example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a factor VIII having a modifiedcalcium binding site, preferably at an amino acid corresponding toresidue 113 of SEQ ID NO: 2. This affords a factor VIIIa having enhancedspecific activity. Exemplary mutants of this type are described in U.S.Patent Application Publ. No. US2007/0265199 to Fay et al., which ishereby incorporated by reference in its entirety. Preferably, theresidue 113 mutant also is modified in accordance with one or more ofthe mutations described above (e.g., at positions 287, 302, 519, 665,and/or 1984) to afford a high stability/high specific activity factorVIII protein. Exemplary high stability/high specific activity factorVIII proteins include, without limitation: those possessing combinedsubstitutions E113AD519A, E113AD519V, E113AE665A, E113AE665V,E113AE1984V, E113AD519AE665V, E113AD519VE665V, E113AD519VE1984A,E113AD519AE665VE1984A, and E113AD519VE665VE1984A.

Examples of mutant factor VIII possessing substitutions at the A1-C2domain interface as well as the residue 113 substitution, and one orboth of the A1-A2 and A2-A3 domain interface substitutions include,without limitation, A108IE113AD519AE665V, A108IE113AD519VE665V,A108IE113AD519VE1984A, A108IE113AD519AE665VE1984A,A108IE113AD519VE665VE1984A, R121C-L2302C/E113AD519AE665V,R121C-L2302C/E113AD519VE665V, R121C-L2302C/E113AD519VE1984A,R121C-L2302C/E113AD519AE665VE1984A, andR121C-L2302C/E113AD519VE665VE1984A. Each of these mutant factor VIII canbe expressed in a B-domainless form, as described below.

A third example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a B-domainless factor VIII thatcontains amino acid residues 1-740 and 1690-2332 of SEQ ID NO: 2 (see,e.g., U.S. Patent No. 6,458,563 to Lollar, which is hereby incorporatedby reference in its entirety).

In one embodiment of the B-domainless recombinant factor VIII of thepresent invention, the B-domain is replaced by a DNA linker segment andat least one codon is replaced with a codon encoding an amino acidresidue that has the same charge as a corresponding residue of porcinefactor VIII (see, e.g., U.S. Patent Application Publication No.2004/0197875 to Hauser et al., which is hereby incorporated by referencein its entirety).

In another embodiment of the B-domainless recombinant factor VIII of thepresent invention, the modified mutant factor VIII is encoded by anucleotide sequence having a truncated factor IX intron 1 inserted inone or more locations (see, e.g., U.S. Pat. No. 6,800,461 to Negrier andU.S. Pat. No. 6,780,614 to Negrier, each of which is hereby incorporatedby reference in its entirety). This recombinant factor VIII can be usedfor yielding higher production of the recombinant factor VIII in vitroas well as in a transfer vector for gene therapy (see, e.g., U.S. Pat.No. 6,800,461 to Negrier, which is hereby incorporated by reference inits entirety). In a particular example of this embodiment, therecombinant factor VIII can be encoded by a nucleotide sequence having atruncated factor IX intron 1 inserted in two locations, and having apromoter that is suitable for driving expression in hematopoietic celllines, and specifically in platelets (see, e.g., U.S. Pat. No. 6,780,614to Negrier, which is hereby incorporated by reference in its entirety).

Regardless of the embodiment, the B-domainless factor VIII preferablycontains one or more of the mutations described above (e.g., modified A1domain interface and/or C2 domain interface, as well as any othermutations to affect other properties of the resulting factor VIII).Recombinant factor VIII proteins prepared in accordance with theExamples herein are B-domainless.

A fourth example of a suitable mutant factor VIII that can be modifiedin accordance with the present invention is a chimeric human/animalfactor VIII that contains one or more domains, or portions thereof, fromhuman factor VIII and one or more domains, or portions thereof, from anon-human mammalian factor VIII. One or more animal amino acid residuescan be substituted for human amino acid residues that are responsiblefor the antigenicity of human factor VIII. In particular, animal (e.g.,porcine) residue substitutions can include, without limitation, one ormore of the following: R484A, R488G, P485A, L486S, Y487L, Y487A, S488A,S488L, R489A, R489S, R490G, L491S, P492L, P492A, K493A, G494S, V495A,K496M, H497L, L498S, K499M, D500A, F501A, P502L, I503M, L504M, P505A,G506A, E507G, I508M, I508A, M2199I, F2200L, L2252F, V2223A, K2227E,and/or L2251 (U.S. Pat. No. 5,859,204 to Lollar, U.S. Pat. No. 6,770,744to Lollar, and U.S. Patent Application Publication No. 2003/0166536 toLollar, each of which is hereby incorporated by reference in itsentirety). Preferably, the recombinant chimeric factor VIII contains oneor more of the mutations described above (e.g., modified A1 domaininterface and/or C2 domain interface).

A fifth example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a factor VIII that has enhancedaffinity for factor IXa (see, e.g., Fay et al., “Factor VIIIa A2 SubunitResidues 558-565 Represent a Factor IXa Interactive Site,” J. Biol.Chem. 269(32):20522-7 (1994); Bajaj et al., “Factor IXa: Factor VIIIaInteraction. Helix 330-338 of Factor IXa Interacts with Residues 558-565and Spatially Adjacent Regions of the A2 Subunit of Factor VIIIa,” J.Biol. Chem. 276(19):16302-9 (2001); and Lenting et al., “The SequenceGlu1811-Lys1818 of Human Blood Coagulation Factor VIII Comprises aBinding Site for Activated Factor IX,” J. Biol. Chem. 271(4):1935-40(1996), each of which is hereby incorporated by reference in itsentirety) and/or factor X (see, e.g., Lapan et al., “Localization of aFactor X Interactive Site in the A1 Subunit of Factor VIIIa,” J. Biol.Chem. 272:2082-88 (1997), which is hereby incorporated by reference inits entirety). Preferably, the enhanced-affinity factor VIII containsone or more of the mutations described above (e.g., modified A1 domaininterface and/or C2 domain interface).

A sixth example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a factor VIII that is modifiedto enhance secretion of the factor VIII (see, e.g., Swaroop et al.,“Mutagenesis of a Potential Immunoglobulin-Binding Protein-Binding SiteEnhances Secretion of Coagulation Factor VIII,” J. Biol. Chem.272(39):24121-4 (1997), which is hereby incorporated by reference in itsentirety). Preferably, the secretion enhanced mutant factor VIIIcontains one or more of the mutations identified above (e.g., modifiedA1 domain interface and/or C2 domain interface).

A seventh example of a suitable mutant factor VIII that can be modifiedin accordance with the present invention is a factor VIII with anincreased circulating half-life. This modification can be made usingvarious approaches, including, without limitation, by reducinginteractions with heparan sulfate (Sarafanov et al., “Cell SurfaceHeparan Sulfate Proteoglycans Participate in Factor VIII CatabolismMediated by Low Density Lipoprotein Receptor-Related Protein,” J. Biol.Chem. 276(15):11970-9 (2001), which is hereby incorporated by referencein its entirety) and/or low-density lipoprotein receptor-related protein(“LRP”) (Saenko et al., “Role of the Low Density Lipoprotein-RelatedProtein Receptor in Mediation of Factor VIII Catabolism,” J. Biol. Chem.274(53):37685-92 (1999); and Lenting et al., “The Light Chain of FactorVIII Comprises a Binding Site for Low Density LipoproteinReceptor-Related Protein,” J. Biol. Chem. 274(34):23734-9 (1999), eachof which is hereby incorporated by reference in its entirety).Preferably, the half-life enhanced mutant factor VIII contains one ormore of the mutations described above (e.g., modified A1 domaininterface and/or C2 domain interface).

An eighth example of a suitable mutant factor VIII that can be modifiedin accordance with the present invention is a modified factor VIIIencoded by a nucleotide sequence modified to code for amino acids withinknown, existing epitopes to produce a recognition sequence forglycosylation at asparagines residues (see, e.g., U.S. Pat. No.6,759,216 to Lollar, which is hereby incorporated by reference in itsentirety). The mutant factor VIII of this example can be useful inproviding a modified factor VIII that escapes detection by existinginhibitory antibodies (low antigenicity factor VIII) and which decreasesthe likelihood of developing inhibitory antibodies (low immunogenicityfactor VIII). In one particular embodiment of this example, the modifiedfactor VIII is mutated to have a consensus amino acid sequence forN-linked glycosylation. An example of such a consensus sequence isN-X-S/T, where N is asparagine, X is any amino acid, and S/T stands forserine or threonine (see U.S. Pat. No. 6,759,216 to Lollar, which ishereby incorporated by reference in its entirety). Preferably, theglycosylation site-modified factor VIII contains one or more of themutations identified above (e.g., modified A1 domain interface and/or C2domain interface).

A ninth example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a modified factor VIII that isa procoagulant-active factor VIII having various mutations (see, e.g.,U.S. Patent Application Publication No. 2004/0092442 to Kaufman et al.,which is hereby incorporated by reference in its entirety). One exampleof this embodiment relates to a modified factor VIII that has beenmodified to (i) delete the von Willebrand factor binding site, (ii) adda mutation at Arg 740, and (iii) add an amino acid sequence spacerbetween the A2- and A3-domains, where the amino acid spacer is of asufficient length so that upon activation, the procoagulant-activefactor VIII protein becomes a heterodimer (see U.S. Patent ApplicationPublication No. 2004/0092442 to Kaufman et al., which is herebyincorporated by reference in its entirety). Preferably, procoagulantactive factor VIII is also modified to contain one or more of themutations described above (e.g., at positions modified A1 domaininterface and/or C2 domain interface).

A tenth example of a suitable mutant factor VIII that can be modified inaccordance with the present invention is a modified factor VIII thatincludes a substitution of one or more amino acid residues within aregion surrounding an activated protein C cleavage site, except that thecleavable Arg scissile bond (at Arg336 and/or Arg562) is not substituted(see, e.g., U.S. Patent Application Publication No. 2009/0118185 to Fayet al., which is hereby incorporated by reference in its entirety). Inthe most preferred embodiments, the one or more substitutions appearswithin the P4-P3′ activated protein C cleavage site, which can be thesite corresponding to wild type residues 333-339 of the A1 domain or thesite corresponding to residues 559-565 of the A2 domain. Exemplarymutant P4-P3′ regions, which include the substitution of one or moreamino acids include, without limitation, VDQRGNQ (SEQ ID NO: 26)neighboring Arg562, VDQRMKN (SEQ ID NO: 27) neighboring Arg562, andPQLRGNQ (SEQ ID NO: 28) neighboring Arg336, PDLRMKN (SEQ ID NO: 29)neighboring Arg336, PQQRMKN (SEQ ID NO: 30) neighboring Arg336, PQRRMKN(SEQ ID NO: 31) neighboring Arg336, PQLRGKN (SEQ ID NO: 32) neighboringArg336, PQLRMIN (SEQ ID NO: 33) neighboring Arg336, and PQLRMNN (SEQ IDNO: 34) neighboring Arg336. These substitutions are preferred forachieving a mutant factor VIIIa having a reduced rate of inactivation byactivated protein C, but unlike mutants having single mutationreplacements of the P1 Arg residue the resulting factor VIIIa is capableof being inactivated by activated protein C. Preferably, factor VIIIhaving a modified activated protein C cleavage site is also modified tocontain one or more of the mutations described above (e.g., at positionsmodified A1 domain interface and/or C2 domain interface).

Further, the mutant factor VIII can be modified to take advantage ofvarious advancements regarding recombinant coagulation factors generally(see, e.g., Saenko et al., “The Future of Recombinant CoagulationFactors,” J. Thrombosis and Haemostasis 1:922-930 (2003), which ishereby incorporated by reference in its entirety).

The recombinant factor VIII of the present invention can be modified atany residue to stabilize the A1/C2 domain interfaces (includes positionscorresponding to 108, 121, 2302, 2328 of the WT factor VIII), as well asbe modified at any charged residue that destabilizes the A1A2 or A2A3domain interfaces (including positions 287, 302, 519, 665, or 1984), bemodified to be B-domainless, to be chimeric, to have modified calciumbinding sites that enhance factor VIIIa activity (e.g., at position113), to have altered inactivation cleavage sites, to have enhancedfactor IXa and/or factor X affinity, to have enhanced secretion, to havean increased circulating half-life, or to have mutant glycosylationsites; or to possess any one or more of such modifications in additionto the one or more modifications to charged residues, including amodified calcium-binding site that improves activity of the recombinantfactor VIII. A number of exemplary B-domainless high stabilityrecombinant factor VIII proteins are described in the Examples.

The recombinant factor VIII is preferably produced in a substantiallypure form. In a particular embodiment, the substantially purerecombinant factor VIII is at least about 80% pure, more preferably atleast 90% pure, most preferably at least 95% pure. A substantially purerecombinant factor VIII can be obtained by conventional techniques wellknown in the art. Typically, the substantially pure recombinant factorVIII is secreted into the growth medium of recombinant host cells.Alternatively, the substantially pure recombinant factor VIII isproduced but not secreted into growth medium. In such cases, to isolatethe substantially pure recombinant factor VIII, the host cell carrying arecombinant plasmid is propagated, lysed by sonication, heat, orchemical treatment, and the homogenate is centrifuged to remove celldebris. The supernatant is then subjected to sequential ammonium sulfateprecipitation. The fraction containing the substantially purerecombinant factor VIII is subjected to gel filtration in anappropriately sized dextran or polyacrylamide column to separate therecombinant factor VIII. If necessary, a protein fraction (containingthe substantially pure recombinant factor VIII) may be further purifiedby high performance liquid chromatography (“HPLC”).

Another aspect of the present invention relates to an isolated nucleicacid molecule that encodes the recombinant factor VIII of the presentinvention. The isolated nucleic acid molecule encoding the recombinantfactor VIII can be either RNA or DNA.

In another embodiment the isolated nucleic acid molecule can have anucleotide sequence encoding a recombinant factor VIII according to thepresent invention which further comprises one or more of (i) factor IXaand/or factor X binding domains modified to enhance the affinity of therecombinant factor VIII for one or both of factor IXa and factor X; (ii)modified sites that enhance secretion in culture; (iii) modified serumprotein binding sites that enhance the circulating half-life thereof;(iv) at least one glycosylation recognition sequence that is effectivein decreasing antigenicity and/or immunogenicity thereof; (v) a modifiedcalcium-binding site that improves specific activity of the recombinantfactor VIIIa; (vi) modified activated protein C-cleavage site; (vii) amodified A1 and A2 domain interface; and (viii) a modified A2 and A3domain interface.

In one embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a modified A1/A2 domain interface or A2/A3domain interface (e.g., at positions corresponding to positions 287,302, 519, 665, 1984 and/or 332-340 of SEQ ID NO: 2), as modified withone or more of the substitutions affecting the A1/C2 domain interfaces(e.g., SEQ ID NOS: 19-25).

In another embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a mutation at position 113 that enhancesfactor VIII specific activity, as modified with one or more of thesubstitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS:19-25).

In another embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a B-domainless factor VIII of the typedescribed above, as modified with one or more of the substitutionsaffecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

In another embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a chimeric human/porcine of the typedescribed above, as modified with one or more of the substitutionsaffecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

In another embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a factor VIII whose inactivation sites havebeen modified as described above, as further modified with one or moreof the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ IDNOS: 19-25).

In yet another embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a factor VIII whose affinity for factor IXaand/or factor X has been enhanced, as further modified with one or moreof the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ IDNOS: 19-25).

In a still further embodiment, the isolated nucleic acid molecule canhave a nucleotide sequence encoding a factor VIII whose affinity forvarious serum-binding proteins has been altered to increase itscirculating half-life, as further modified with one or more of thesubstitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS:19-25).

In a further embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a factor VIII that has increased secretionin culture, as further modified with one or more of the substitutionsaffecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

In a further embodiment, the isolated nucleic acid molecule can have anucleotide sequence encoding a factor VIII that possesses one or morenon-naturally occurring glycosylation site, as further modified with oneor more of the substitutions affecting the A1/C2 domain interfaces(e.g., SEQ ID NOS: 19-25).

In a still further embodiment, the isolated nucleic acid molecule canhave a nucleotide sequence encoding a factor VIII that has a modifiedactivated protein C cleavage site, as further modified with one or moreof the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ IDNOS: 19-25).

In yet another embodiment, the isolated nucleic acid molecule encodes arecombinant factor VIII that is modified at any one or more chargedresidues as described above and is also modified to possess any two ormore of the following: modified to be B-domainless, modified to bechimeric, modified to have altered inactivation cleavage sites, modifiedto have enhanced factor IXa and/or factor X affinity, modified to haveenhanced secretion, modified to have an increased circulating half-life,modified to possess one or more non-naturally occurring glycosylationsite, modified within a calcium-binding site (e.g., at position 113)such that the specific activity of the recombinant factor VIII isimproved, modified activated protein C-cleavage site, a modified A1 andA2 domain interface, and a modified A2 and A3 domain interface.

Another aspect of the present invention relates to a recombinant DNAexpression system that includes an isolated DNA molecule of the presentinvention, which expression system encodes a recombinant factor VIII. Inone embodiment, the DNA molecule is in sense orientation relative to apromoter.

A further aspect of the present invention relates to a host cellincluding an isolated nucleic acid molecule encoding the recombinantfactor VIII of the present invention. In a particular embodiment, thehost cell can contain the isolated nucleic acid molecule in DNA moleculeform, either as a stable plasmid or as a stable insertion or integrationinto the host cell genome. In another embodiment, the host cell cancontain a DNA molecule in an expression system. Suitable host cells canbe, without limitation, animal cells (e.g., baby hamster kidney (“BHK”)cells), bacterial cells (e.g., E. coli), insect cells (e.g., Sf9 cells),fungal cells, yeast cells (e.g., Saccharomyces or Schizosaccharomyces),plant cells (e.g., Arabidopsis or tobacco cells), or algal cells.

The recombinant DNA expression system and host cells can be producedusing various recombinant techniques well-known in the art, as furtherdiscussed below.

The DNA molecule encoding the recombinant factor VIII of the presentinvention can be incorporated in cells using conventional recombinantDNA technology. Generally, this involves inserting the DNA molecule intoan expression system to which the DNA molecule is heterologous (i.e.,not normally present). The heterologous DNA molecule is inserted intothe expression system or vector in sense orientation and correct readingframe. The vector contains the necessary elements for the transcriptionand translation of the inserted protein-coding sequences. Thus, oneembodiment of the present invention provides a DNA construct containingthe isolated nucleic acid of the present invention, which is operablylinked to both a 5′ promoter and a 3′ regulatory region (i.e.,transcription terminator) capable of affording transcription andexpression of the encoded recombinant factor VIII of the presentinvention in host cells or host organisms.

With respect to the recombinant expression system of the presentinvention, an expression vector containing a DNA molecule encoding therecombinant factor VIII of the present invention can be made usingcommon techniques in the art. The nucleic acid molecules of the presentinvention can be inserted into any of the many available expressionvectors using reagents that are well known in the art. In preparing aDNA vector for expression, the various DNA sequences may normally beinserted or substituted into a bacterial plasmid. Any convenient plasmidmay be employed, which will be characterized by having a bacterialreplication system, a marker which allows for selection in a bacterium,and generally one or more unique, conveniently located restrictionsites. The selection of a vector will depend on the preferredtransformation technique and target host for transformation.

A variety of host-vector systems may be utilized to express therecombinant factor VIII-encoding sequence(s). Primarily, the vectorsystem must be compatible with the host cell used. Host-vector systemsinclude but are not limited to the following: bacteria transformed withbacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such asyeast containing yeast vectors; mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, adeno-associated virus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria (e.g., Agrobacterium). The expressionelements of these vectors vary in their strength and specificities.Depending upon the host-vector system utilized, any one of a number ofsuitable transcription and translation elements can be used.

When recombinantly produced, the factor VIII protein or polypeptide (orfragment or variant thereof) is expressed in a recombinant host cell,typically, although not exclusively, a eukaryote.

Suitable vectors for practicing the present invention include, but arenot limited to, the following viral vectors such as lambda vector systemgt11, gtWES.tB, Charon 4, and plasmid vectors such as pCMV, pBR322,pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/−(see “StratageneCloning Systems” Catalog (1993)), pQE, pIH821, pGEX, pET series (Studieret al, “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,”Methods in Enzymology 185:60-89 (1990), which is hereby incorporated byreference in its entirety), and any derivatives thereof. Any appropriatevectors now known or later described for genetic transformation aresuitable for use with the present invention.

Recombinant molecules can be introduced into cells via transformation,particularly transduction, conjugation, mobilization, orelectroporation. The DNA sequences are cloned into the vector usingstandard cloning procedures in the art, as described by Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, N.Y.: ColdSprings Laboratory, (1982), which is hereby incorporated by reference inits entirety.

U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is herebyincorporated by reference in its entirety, describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase. These recombinant plasmidsare then introduced by means of transformation and replicated inunicellular cultures including prokaryotic organisms and eukaryoticcells grown in tissue culture.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eukaryotic promotersdiffer from those of prokaryotic promoters. Furthermore, eukaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a prokaryotic system, and, further, prokaryoticpromoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper prokaryotic signals which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology 68:473 (1979), which ishereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isgenerally desirable to use strong promoters in order to obtain a highlevel of transcription and, hence, expression of the gene. Dependingupon the host cell system utilized, any one of a number of suitablepromoters may be used. For instance, when cloning in Escherichia coli,its bacteriophages, or plasmids, promoters such as the T7 phagepromoter, lac promoter, trp promoter, recA promoter, ribosomal RNApromoter, the P_(R) and P_(L) promoters of coliphage lambda and others,including but not limited, to lacUV5, ompF, bla, lpp, and the like, maybe used to direct high levels of transcription of adjacent DNA segments.Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. colipromoters produced by recombinant DNA or other synthetic DNA techniquesmay be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

In one embodiment, the nucleic acid molecule of the present invention isincorporated into an appropriate vector in the sense direction, suchthat the open reading frame is properly oriented for the expression ofthe encoded protein under control of a promoter of choice. This involvesthe inclusion of the appropriate regulatory elements into the DNA-vectorconstruct. These include non-translated regions of the vector, usefulpromoters, and 5′ and 3′ untranslated regions which interact with hostcellular proteins to carry out transcription and translation. Suchelements may vary in their strength and specificity. Depending on thevector system and host utilized, any number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, may be used.

A constitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed.

The DNA construct of the present invention can also include an operable3′ regulatory region, selected from among those which are capable ofproviding correct transcription termination and polyadenylation of mRNAfor expression in the host cell of choice, operably linked to a DNAmolecule which encodes for a protein of choice.

The vector of choice, promoter, and an appropriate 3′ regulatory regioncan be ligated together to produce the DNA construct of the presentinvention using well known molecular cloning techniques as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. CurrentProtocols in Molecular Biology, New York, N.Y: John Wiley & Sons (1989),each of which is hereby incorporated by reference in its entirety.

As noted, one alternative to the use of prokaryotic host cells is theuse of eukaryotic host cells, such as mammalian cells, which can also beused to recombinantly produce the recombinant factor VIII of the presentinvention. Mammalian cells suitable for carrying out the presentinvention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651),BHK (e.g., ATCC No. CRL 6281), CHO (e.g., ATCC No. CCL 61), HeLa (e.g.,ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells.

Suitable expression vectors for directing expression in mammalian cellsgenerally include a promoter, as well as other transcription andtranslation control sequences known in the art. Common promoters includeSV40, MMTV, metallothionein-1, adenovirus E1a, CMV, immediate early,immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.

Once the DNA construct of the present invention has been prepared, it isready to be incorporated into a host cell. Accordingly, another aspectof the present invention relates to a method of making a recombinantcell. Basically, this method is carried out by transforming a host cellwith a DNA construct of the present invention under conditions effectiveto yield transcription of the DNA molecule in the host cell. Recombinantmolecules can be introduced into cells via transformation, particularlytransduction, conjugation, mobilization, or electroporation.

In view of the recombinant technology discussed herein, another aspectof the present invention relates to a method of making a recombinantfactor VIII of the present invention. This method involves growing ahost cell of the present invention under conditions whereby the hostcell expresses the recombinant factor VIII of the present invention. Therecombinant factor VIII is then isolated. In one embodiment, the hostcell is grown in vitro in a growth medium. In a particular embodiment,suitable growth media can include, without limitation, a growth mediumcontaining a von Willebrand Factor (referred to herein as “VWF”). Inthis embodiment, the host cell can contain a transgene encoding a VWF orthe VWF can be introduced to the growth medium as a supplement. VWF inthe growth medium will allow for greater expression levels of therecombinant factor VIII. Once the recombinant factor VIII is secretedinto the growth medium, it can then be isolated from the growth mediumusing techniques well-known by those of ordinary skill in the relevantrecombinant DNA and protein arts (including those described herein). Inanother embodiment, the method of making the recombinant factor VIII ofthe present invention further involves disrupting the host cell prior toisolation of the recombinant factor VIII. In this embodiment, therecombinant factor VIII is isolated from cellular debris.

The modifications at positions corresponding to 108, 121/2302, 2328 ofthe WT factor VIII are particularly preferred, because they result inenhanced stability of factor VIII and, when used in combination withmodifications at residues 519, 665, and/or 1984, result in significantlyenhanced stability of both factor VIII and factor VIIIa. This increasedstability is important with regard to circulating half-life of factorVIII and the activity of factor VIIIa during blood clotting.Furthermore, this property is significant in terms of enhancing therecovery of usable factor VIII during the purification and preparationof the protein for therapeutic use, particularly given the improvedthermal and chemical stability of factor VIII.

When an expression vector is used for purposes of in vivo transformationto induce factor VIII expression in a target cell, promoters of varyingstrength can be employed depending on the degree of enhancement desired.One of skill in the art can readily select appropriate mammalianpromoters based on their strength as a promoter. Alternatively, aninducible promoter can be employed for purposes of controlling whenexpression or suppression of factor VIII is desired. One of skill in theart can readily select appropriate inducible mammalian promoters fromthose known in the art. Finally, tissue specific mammalian promoters canbe selected to restrict the efficacy of any gene transformation systemto a particular tissue. Tissue specific promoters are known in the artand can be selected based upon the tissue or cell type to be treated.

Another aspect of the present invention relates to a method of treatingan animal for a blood disorder such as hemophilia, particularlyhemophilia A. This method involves administering to an animal exhibitinghemophilia A an effective amount of the recombinant factor VIII of thepresent invention, whereby the animal exhibits effective blood clottingfollowing vascular injury. A suitable effective amount of therecombinant factor VIII can include, without limitation, between about10 to about 50 units/kg body weight of the animal. The animal can be anymammal, but preferably a human, a rat, a mouse, a guinea pig, a dog, acat, a monkey, a chimpanzee, an orangutan, a cow, a horse, a sheep, apig, a goat, or a rabbit.

The recombinant factor VIII of the present invention can be used totreat uncontrolled bleeding due to factor VIII deficiency (e.g.,intraarticular, intracranial, or gastrointestinal hemorrhage) inhemophiliacs with and without inhibitory antibodies and in patients withacquired factor VIII deficiency due to the development of inhibitoryantibodies. In a particular embodiment, the recombinant factor VIII,alone, or in the form of a pharmaceutical composition (i.e., incombination with stabilizers, delivery vehicles, and/or carriers) isinfused into patients intravenously according to the same procedure thatis used for infusion of human or animal factor VIII.

Alternatively, or in addition thereto, the recombinant factor VIII canbe administered by administering a viral vector such as anadeno-associated virus (Gnatenko et al., “Human Factor VIII Can BePackaged and Functionally Expressed in an Adeno-associated VirusBackground: Applicability to Hemophilia A Gene Therapy,” Br. J.Haematol. 104:27-36 (1999), which is hereby incorporated by reference inits entirety), or by transplanting cells genetically engineered toproduce the recombinant factor VIII, typically via implantation of adevice containing such cells. Such transplantation typically involvesusing recombinant dermal fibroblasts, a non-viral approach (Roth et al.,“Nonviral Transfer of the Gene Encoding Coagulation Factor VIII inPatients with Sever Hemophilia,” New Engi. J. Med. 344:1735-1742 (2001),which is hereby incorporated by reference in its entirety).

The treatment dosages of recombinant factor VIII that should beadministered to a patient in need of such treatment will vary dependingon the severity of the factor VIII deficiency. Generally, dosage levelis adjusted in frequency, duration, and units in keeping with theseverity and duration of each patient's bleeding episode. Accordingly,the recombinant factor VIII is included in a pharmaceutically acceptablecarrier, delivery vehicle, or stabilizer in an amount sufficient todeliver to a patient a therapeutically effective amount of the proteinto stop bleeding, as measured by standard clotting assays.

Factor VIII is classically defined as that substance present in normalblood plasma that corrects the clotting defect in plasma derived fromindividuals with hemophilia A. The coagulant activity in vitro ofpurified and partially-purified forms of factor VIII is used tocalculate the dose of recombinant factor VIII for infusions in humanpatients and is a reliable indicator of activity recovered from patientplasma and of correction of the in vivo bleeding defect. There are noreported discrepancies between standard assay of novel factor VIIImolecules in vitro and their behavior in the dog infusion model or inhuman patients, according to Lusher et al., “Recombinant Factor VIII forthe Treatment of Previously Untreated Patients with Hemophilia A—Safety,Efficacy, and Development of Inhibitors,” New Engl. J. Med. 328:453-459(1993); Pittman et al., “A2 Domain of Human Recombinant-derived FactorVIII is Required for Procoagulant Activity but not for ThrombinCleavage,” Blood 79:389-397 (1992); and Brinkhous et al., “PurifiedHuman Factor VIII Procoagulant Protein Comparative Hemostatic ResponseAfter Infusions into Hemophilic and von Willebrand Disease Dogs,” Proc.Natl. Acad. Sci. 82:8752-8755 (1985), which are hereby incorporated byreference in their entirety.

Usually, the desired plasma factor VIII activity level to be achieved inthe patient through administration of the recombinant factor VIII is inthe range of 30-100% of normal. In one embodiment, administration of thetherapeutic recombinant factor VIII is given intravenously at apreferred dosage in the range from about 5 to 50 units/kg body weight,and particularly in a range of 10-50 units/kg body weight, and furtherparticularly at a dosage of 20-40 units/kg body weight; the intervalfrequency is in the range from about 8 to 24 hours (in severely affectedhemophiliacs); and the duration of treatment in days is in the rangefrom 1 to 10 days or until the bleeding episode is resolved. See, e.g.,Roberts and Jones, “Hemophilia and Related Conditions—CongenitalDeficiencies of Prothrombin (Factor II, Factor V, and Factors VII toXII),” Ch. 153, 1453-1474, 1460, in Hematology, Williams, W. J., et al.,ed. (1990), which is hereby incorporated by reference in its entirety.Patients with inhibitors may require a different amount of recombinantfactor VIII than their previous form of factor VIII. For example,patients may require less recombinant factor VIII because of its higherspecific activity than the wild-type VIII and its decreased antibodyreactivity. As in treatment with human or plasma-derived factor VIII,the amount of therapeutic recombinant factor VIII infused is defined bythe one-stage factor VIII coagulation assay and, in selected instances,in vivo recovery is determined by measuring the factor VIII in thepatient's plasma after infusion. It is to be understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed recombinant factor VIII.

Treatment can take the form of a single intravenous administration ofthe recombinant factor VIII or periodic or continuous administrationover an extended period of time, as required. Alternatively, therapeuticrecombinant factor VIII can be administered subcutaneously or orallywith liposomes in one or several doses at varying intervals of time.

The recombinant factor VIII can also be used to treat uncontrolledbleeding due to factor VIII deficiency in hemophiliacs who havedeveloped antibodies to human factor VIII.

It has been demonstrated herein that the recombinant factor VIII of thepresent invention can differ in specific activity from the wild-typefactor VIII and retain a higher specific activity for a longer durationfollowing activation. Factor VIII proteins having greater procoagulantactivity from wild-type factor VIII are useful in treatment ofhemophilia because lower dosages will be required to correct a patient'sfactor VIII deficiency. This will not only reduce medical expense forboth the patient and the insurer, but also reduce the likelihood ofdeveloping an immune response to the factor VIII (because less antigenis administered).

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention, but they are by no means intended to limit its scope.

Materials & Methods

Reagents:

Recombinant factor VIII (KOGENATE™) and the monoclonal antibodies 58.12and 2D2 were generous gifts from Dr. Lisa Regan of Bayer Corporation(Berkeley, Calif.). Phospholipid vesicles containing 20%phosphatidylcholine (PC), 40% phosphatidylethanolamine (PE), and 40%phosphatidylserine (PS) were prepared using octylglucoside as describedpreviously (Mimms et al., “Phospholipid Vesicle Formation andTransmembrane Protein Incorporation Using Octyl Glucoside,” Biochemistry20:833-840 (1981), which is hereby incorporated by reference in itsentirety). The reagents α-thrombin, factor VIIa, factor IXaβ, factor X,and factor Xa (Enzyme Research Laboratories, South Bend, Ind.), hirudin(DiaPharma, West Chester, Ohio), phospholipids (Avanti Polar Lipids,Alabaster, Ala.), the chromogenic Xa substrate, Pefachrome Xa(Pefa-5523, CH₃OCO-D-Cha-Gly-Arg-pNA.AcOH; Centerchem Inc. NorwalkConn.), recombinant human tissue factor (rTF), Innovin (Dade Behring,Newark, Del.), fluorogenic substrate, Z-Gly-Gly-Arg-AMC (Calbiochem, SanDiego, Calif.), thrombin calibrator (Diagnostica Stago, Parsippany,N.J.), and acrylodan (Molecular Probes, Eugene, Oreg.) were purchasedfrom the indicated vendors.

Expression and Purification of WT and Variant Factor VIII:

Recombinant WT and variant factor VIII forms were stably expressed inBHK cells and purified as described previously (Wakabayashi et al.,“Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca²⁺ BindingSite Required for Cofactor Activity,” J Biol. Chem. 279:12677-12684(2004), which is hereby incorporated by reference in its entirety).After transfection there were no significant differences in the amountsof factor VIII secretion among the variants. Protein yields for thevariants ranged from >10 to ˜100 μg from two 750 cm² culture flasks,with purity from ˜85% to >95% as judged by SDS-PAGE. The primarycontaminant in the factor VIII preparations was albumin and at theconcentrations present in the factor VIII showed no effect on stabilityor activity parameters. Factor VIII concentration was measured by ELISAand factor VIII activity was determined by one-stage clotting assay andtwo-stage chromogenic factor Xa generation assay, both of which aredescribed below.

Western Blotting:

Factor VIII proteins (0.34 μg) were activated by thrombin (20 nM) for 30min at 23° C. and subjected to electrophoresis under either non-reducingor reducing (0.1 M dithiothreitol) conditions using 10% polyacrylamidegels run at constant voltage (150V). Gels were transferred to apolyvinylidene fluoride membrane, probed with an anti-A1 domain (58.12)or anti-A3 domain (2D2) monoclonal antibody and protein bands werevisualized using chemifluorescence. The chemifluorescence substrate (ECFsubstrate, GE Healthcare, Piscataway, N.J.) was reacted and thefluorescence signal scanned using a phosphoimager (Storm 860, GEHealthcare).

ELISA:

A sandwich ELISA was performed to measure the concentration of factorVIII proteins as previously described (Wakabayashi et al., “A Glu113AlaMutation within a Factor VIII Ca²⁺-Binding Site Enhances CofactorInteractions in Factor Xase,” Biochemistry 44:10298-10304 (2005), whichis hereby incorporated by reference in its entirety) using purifiedcommercial recombinant factor VIII (KOGENATE™, Bayer Corporation) as astandard. Factor VIII capture used the anti-C2 monoclonal antibody(GMA8003, Green Mountain Antibodies) and the anti-A2 monoclonalantibody, R8B12 (GMA8012, Green Mountain Antibodies) was employed forfactor VIII detection following biotinylation.

One-stage Clotting Assay:

One-stage clotting assays were performed using substrate plasmachemically depleted of factor VIII (Over, “Methodology of the One-stageAssay of Factor VIII (VIII:C),” Scand J Haematol Suppl. 41:13-24 (1984),which is hereby incorporated by reference in its entirety) and assayedusing a Diagnostica Stago clotting instrument. Plasma was incubated withAPTT reagent (General Diagnostics) for 6 min at 37° C. after which adilution of factor VIII was added to the cuvette. After 1 min themixture was recalcified, and clotting time was determined and comparedto a pooled normal plasma standard.

Two-stage Chromogenic Factor Xa Generation Assay:

The rate of conversion of factor X to factor Xa was monitored in apurified system (Lollar et al., “Factor VIII and Factor VIIIa,” MethodsEnzymol. 222:128-143 (1993), which is hereby incorporated by referencein its entirety) according to methods previously described (Wakabayashiet al., “Metal Ion-independent Association of Factor VIII Subunits andthe Roles of Calcium and Copper Ions for Cofactor Activity andInter-subunit Affinity,” Biochemistry 40:10293-10300 (2001); Wakabayashiet al., “Ca²⁺ Binding to Both the Heavy and Light Chains of Factor VIIIIs Required for Cofactor Activity,” Biochemistry 41:8485-8492 (2002),each of which is hereby incorporated by reference in its entirety).Factor VIII (1 nM) in buffer containing 20 mMN-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), pH 7.2,0.1 M NaCl, 0.01% Tween 20, 0.01% BSA, 5 mM CaCl₂ (Buffer B), and 10 μMPSPCPE vesicles was activated with 20 nM α-thrombin for 1 min. Thereaction was stopped by adding hirudin (10 U/ml) and the resultingfactor VIIIa was reacted with factor IXa (40 nM) for 1 min. Factor X(300 nM) was added to initiate reactions which were quenched after 1 minby the addition of 50 mM EDTA. Factor Xa generated was determinedfollowing reaction with the chromogenic substrate Pefachrome Xa (0.46 mMfinal concentration). All reactions were run at 23° C.

Factor VIII Activity at Elevated Temperature:

WT factor VIII or factor VIII variants (4 nM) in buffer B were incubatedat 57° C. (pH at this temperature=6.94). Aliquots were removed at theindicated times, cooled to room temperature, and residual factor VIIIactivity was determined using a two-stage factor Xa generation assay.

Factor VIIIa Activity Decay:

WT and mutant factor VIII (1.5 nM) in buffer B containing 20 μM PSPCPEvesicles were activated using 20 nM thrombin for 1 min at 23° C.Reactions were immediately quenched by hirudin (10 U/ml) to inactivatethrombin, aliquots removed at the indicated times, and activity wasdetermined using the factor Xa generation assay following addition offactor IXa (40 nM) and factor X (300 nM).

Factor VIII Activity Inhibition by Guanidinium Chloride:

WT and factor VIII variants (50 nM) in buffer B plus 0-1.8 M guanidiniumchloride were incubated for 2 hrs at 23° C. Aliquots were diluted (1/50)in buffer A containing 20 μM PSPCPE vesicles and activated by 5 nMthrombin for 1 min. Reactions were immediately quenched with hirudin (10U/ml) and activity was determined by factor Xa generation assayfollowing addition of factor IXa (40 nM) and FX (300 nM). Residualguanidinium chloride (<36 mM) did not inhibit the proteolytic activationof factor VIII or its cofactor activity.

Thermal Denaturation of Reconstituted A1 and A3C1C2 or A3C1 Subunit asDetected by FXa Generation Assay—

A1 subunit (50 nM) from WT or Ala108Ile factor VIII was reconstitutedwith A3C1C2 (200 nM) or A3C1 (500 nM) at 37° C. for 2 hr in 10 mM MES,pH 6.5, 0.15 M NaCl, 0.01% Tween 20, 0.01% BSA, 5 mM CaCl₂. Samples wereincubated at 55° C. (A3C1C2) or 52° C. (A3C1) (pH at thistemperature=6.94), aliquots were taken at indicated times, and furtherincubated with 200 nM A2 subunit at 23° C. for 30 min. Samples were thendiluted 1:20 with buffer B containing 20 μM PSPCPE vesicles andreconstituted factor VIIIa activity was measured directly by factor Xageneration assay in the absence of the thrombin activation step. Datawere fitted to the single exponential decay equation by non-linear leastsquares regression and parameter values were obtained.

Thrombin Generation Assay—

The amount of thrombin generated in plasma was measured by CalibratedAutomated Thrombography using methods previously described (Wakabayashiet al., “Combining Mutations of Charged Residues at the A2 DomainInterface Enhances Factor VIII Stability over Single Point Mutations,”J. Thromb. Haemost. 7:438-444 (2009), which is hereby incorporated byreference in its entirety). Briefly, factor VIII deficient plasma (<1%residual activity, platelet-poor) from severe hemophilia A patientslacking factor VIII inhibitor (George King Bio-Medical, Overland Park,Kans.) was mixed at 37° C. with a final concentration of 0.3 nM factorVIII, 0.5 μM rTF, 4 μM PSPCPE vesicles, 433 μM fluorogenic substrate,13.3 mM CaCl₂, and 105 nM thrombin calibrator. The development of afluorescent signal was monitored at 8 second intervals using aMicroplate Spectrofluorometer (Spectramax Gemini, Molecular Devices,Sunnyvale, Calif.) with a 355 nm (excitation)/460 nm (emission) filterset. Fluorescent signals were corrected by the reference signal from thethrombin calibrator samples and actual thrombin generation in nM wascalculated.

Data Analysis:

For activity decay analysis of factor VIII/VIIIa, activity values as afunction of time were fitted to a single exponential decay curve bynon-linear least squares regression using the equation,

A=A ₀ ·e ^(−k·t)

where A is residual factor VIIIa activity (nM/min/nM factor VIII), A₀ isthe initial activity, k is the apparent rate constant, and t is the time(minutes) of reaction of factor VIII (for factor VIII thermal decayexperiments) or of factor VIIIa after thrombin was quenched (for factorVIIIa decay measurements). Factor VIII activity inhibition byguanidinium was fitted to a linear equation by least squares regressionusing the equation,

A=50−k·(X−IC ₅₀)

where A is the normalized activity [=100(%)], IC₅₀ is the inhibitor(guanidinium chloride) concentration (M) at 50% activity, X is theguanidinium chloride concentration (M), and k is the slope.Determinations for A1-A3C1C2 binding affinity used the quadraticequation:

$F = {\frac{F_{\max}}{B} \cdot \frac{\left( {B + K_{d} + X} \right)^{2} - \sqrt{\left( {B + K_{d} + X} \right)^{2} - {4 \cdot B \cdot X}}}{2}}$

where F_(max) is the maximum increase in fluorescence at saturation, Bis the A1 concentration (=15 nM), K_(d) is the dissociation constant,and X is the concentration of A3C1C2 in nM, Nonlinear least-squaresregression analysis was performed using Kaleidagraph (Synergy, Reading,Pa.). A Student's t-test was performed for statistical analysis.

Example 1 Recombinant Expression and Purification of Factor VIII MutantsPossessing Modified A1 and C2 Domain Interactions

The A1 and C2 domains show close proximity to one another in the factorVIII crystal structure (FIG. 3A). These regions were investigated withthe aim towards enhancing inter-domain interactions to positively alteraffinity and stability parameters. Examination of the A1-C2 interfaceprovided two possible approaches for increasing the inter-domainaffinity. Although the orientation of side chains cannot be discerneddue to the resolution (˜4 Å) of the structure (4), the putative spatialseparation of several paired residues indicated that mutagenesis ofthese residues to Cys could result in formation of a nascentinter-domain disulfide bridge. Paired A1/C2 domain residues wereidentified that appeared to meet this distance requirement and includedSer119/Pro2300, Gln120/Pro2299, Arg121/Leu2302, Ala108/Ala2328, andTrp106/Ala2328.

Double mutations where each residue of the respective pair was replacedwith Cys were prepared as B-domainless factor VIII—lacking residuesGln744-Ser1637 in the B-domain (Doering et al., “Expression andCharacterization of Recombinant Murine Factor VIII,” Thromb. Haemost88:450-458 (2002), which is hereby incorporated by reference in itsentirety)—using previously described methods (Wakabayashi et al.,“Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca²⁺-BindingSite Required for Cofactor Activity,” J. Biol. Chem. 279:12677-12684(2004), which is hereby incorporated by reference in its entirety).Briefly, B-domainless factor VIII cDNA was restricted from the factorVIII expression construct HSQ-MSAB-NotI-RENeo using the endonucleasesXhoI and NotI, and then cloned into the Bluescript II K/S-vector. FactorVIII molecules bearing one or more point mutations were constructed byintroducing mutations into shuttle constructs using the StratageneQuikChange site-directed mutagenesis kit. Upon confirmation of thepresence of only the desired mutations by sequencing, the appropriatefragment was restricted and cloned back into the factor VIII expressionconstruct. Presence of only the desired mutations was again confirmed bya second round of sequencing. Of the five variants examined, only theArg121Cys/Leu2302Cys variant (FIG. 3B) retained a wild-type likespecific activity (˜86% the WT value, Table 1) and was furtherevaluated.

Recombinant WT and variant factor VIII forms were stably expressed inBHK cells and purified as previously described (Lollar and Parker,“pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII,”J. Biol. Chem. 265:1688-1692 (1990), which is hereby incorporated byreference in its entirety). Protein yields for the variants rangedfrom >10 to ˜100 μg from two 750 cm² culture flasks, with purity >90% asjudged by SDS-PAGE. The primary contaminant in the factor VIIIpreparations was albumin. Factor VIII concentrations were measured usingan Enzyme-Linked Immunoadsorbant Assay (ELISA) and factor VIII activitywas determined by one-stage clotting and two-stage chromogenic factor Xageneration assays described supra.

In addition, a small cavity in the A1-C2 interface that potentiallyexists was noted, this putative cavity is mainly surrounded by alkylgroups from Ala108, Ala2328, Leu2302, and Gln2329 (FIG. 3C), with theside chain of Ala108 proposed to be directed towards the C2 domain. Inan effort to increase hydrophobic interactions within this area, severalvariants were prepared to introduce bulky hydrophobic groups in thesedomains. This was carried out by mutating Ala residues to Ile, Leu, andVal (Ala108Ile, Ala108Leu, Ala108Val, Ala2328Ile, Ala2328Leu, andAla2328Val) in a B-domainless factor VIII cDNA using the proceduresdescribed in the preceding paragraph. Of the variants prepared, theAla108Ile factor VIII variant showed minimal effects on factor VIIIspecific activity (˜74% the WT value, Table 1); the Ala108Leu andAla108Val variants showed greater diminution of factor VIII specificactivity.

The A1 subunit was purified from WT or Ala108Ile factor VIII. FactorVIII (1-3 μM) was reacted with thrombin (50 nM) in 20 mM HEPES, pH 7.2,0.1 M NaCl, 0.01% Tween 20 (buffer A) for 30 min and treated with 50 mMEDTA overnight at 4° C. After a 1:4 dilution with buffer A, the sampleswere subjected to chromatography using a heparin Sepharose column (1.5cm×0.7 cm in diameter, GE Healthcare, Piscataway, N.J.). The flowthrough fraction was collected and applied to a Q-Sepharose column (1.5cm×0.7 cm in diameter, GE Healthcare). After the column was washed withbuffer A, bound A1 subunit was eluted with 20 mM HEPES, pH 7.2, 0.8 MNaCl, 0.01% Tween20, and purified A1 subunit was kept frozen at 80° C.until use. A2 and A3C1C2 subunits were completely absorbed by theheparin Sepharose column step and the final A1 product was >95% pure asjudged by SDS-PAGE. A2 and A3C1C2 subunits were purified fromrecombinant factor VIII (Kogenate™) as described previously (Fay andSmudzin, “Characterization of the Interaction Between the A2 Subunit andA1/A3-C1-C2 Dimer in Human Factor VIIIa,” J. Biol. Chem. 267:13246-13250(1992), which is hereby incorporated by reference in its entirety). A3C1subunit was purified from C2 domain-deleted factor VIII (Wakabayashi etal., “Factor VIII Lacking the C2 Domain Retains Cofactor Activity invitro,” J. Biol. Chem. 285:25176-25184 (2010), which is herebyincorporated by reference in its entirety) using the same method forA3C1C2 purification.

Purified A1 subunit from WT and Ala108Ile factor VIII was labeled withacrylodan by sulfhydryl specific protein modification as previouslydescribed (Wakabayashi et al., “Metal Ion-independent Association ofFactor VIII Subunits and the Roles of Calcium and Copper Ions forCofactor Activity and Inter-subunit Affinity,” Biochemistry40:10293-10300 (2001), which is hereby incorporated by reference in itsentirety). A1 (15 nM) from WT or Ala108Ile factor VIII was reconstitutedwith A3C1C2 subunit (0-300 nM) at 37° C. for 2 h in buffer B at pH 7.4.Fluorescence measurements were performed using an Aminco-Bowman Series 2Luminescence Spectrometer (Thermo Spectronic, Rochester, N.Y.) at 23° C.at an excitation wavelength of 395 nm (2 nm bandwidth). Fluorescenceemission was monitored at 480-490 nm (8 nm bandwidth) and all spectrawere corrected for background. Data were fitted to a quadratic equationby non-linear least squares regression and parameter values wereobtained.

Example 2 Confirmation of Disulfide Bridge in Arg121Cys/Leu2302CysFactor VIII Variant

Evidence for high efficiency disulfide bridging between factor VIII A1and A3C1C2 domains in this double mutant, as judged by Western Blottingis shown in FIG. 4. For this analysis, WT factor VIII and theArg121Cys/Leu2302Cys factor VIII variant were cleaved with thrombin togenerate the factor VIIIa heterotrimer prior to SDS-PAGE, which was thenrun in the absence and presence of disulfide bond reduction using DTT.Blots were probed with an anti-A1 antibody (58.12, lanes 1-4) and ananti-A3 antibody (2D2, lanes 5-8). Both A1 and A3C1C2 subunits derivedfrom the factor VIII Arg121Cys/Leu2302Cys variant were detected at the120 kDa band, consistent with the sum of their mol masses undernon-reducing conditions (lanes 2 and 6), while reduction by 0.1 M DTTyielded the separated subunits (lanes 4 and 8). Based upon the banddensities of bridged and free subunits in the non-reduced lanes, itappeared that >90% of the variant molecules were disulfide-linked.

Example 3 Affinity of the WT and Ala108Ile Factor VIII Variant A1Subunits for A3C1C2

To assess the affinity of the Ala108Ile A1 domain for C2 domain, thepurified factor VIII variant and WT were treated with thrombin and theA1 subunits were separately purified as described supra. A1 subunitswere then reacted with the environment-sensitive fluorescent probe,acrylodan, and these reagents were used to probe binding with the A3C1C2subunit. The site for acrylodan attachment is likely the lone free thiolin A1 at Cys310, which is in close proximity (<15 Å) to residues in theA3 domain of light chain (Ngo et al., “Crystal Structure of Human FactorVIII: Implications for the Formation of the Factor IXa:Factor VIIIaComplex,” Structure 16:597-606 (2008), which is hereby incorporated byreference in its entirety). Indeed, increases in the emissionfluorescence from acrylodan-labeled A1 (AcA1) subunit have beenpreviously observed when A3C1C2 was bound to the molecule (Wakabayashiet al, “Metal-ion Independent Factor VIII Subunit Association and theRole of Calcium and Copper for Its Affinity and Activity,” Biochemistry40:10293-10300 (2001); Ansong et al., “Factor VIII A3 Domain Residues1954-1961 Represent an A1 Domain-Interactive Site,” Biochemistry44:8850-8857 (2005), each of which is hereby incorporated by referencein its entirety). Titration of AcA1 with A3C1C2 was performed asdescribed supra and the results are shown in FIG. 5. AcA1 (15 nM)fluorescence from both the WT and Ala108Ile subunits saturably increasedas the A3C1C2 concentration increased. The estimated K_(d) of thisinteraction for WT and Ala108Ile A1 subunits were 88.7±9.8 nM and24.1±4.1 nM, respectively. The K_(d) value for WT was somewhat higherthan a previously reported value (˜50 nM) (Ansong et al., “Factor VIIIA3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site,”Biochemistry 44:8850-8857 (2005), which is hereby incorporated byreference in its entirety) likely due to slightly higher pH (7.4)employed for the binding conditions (Wakabayashi et al., “pH-dependentAssociation of Factor VIII Chains: Enhancement of Affinity atPhysiological pH by Cu²⁺ ,” Biochim. Biophys. Acta 1764:1094-1101(2006), which is hereby incorporated by reference in its entirety). Thisresult indicated a ˜4-fold increase in affinity of Ala108Ile A1 forA3C1C2 as compared with the WT A1 subunit for A3C1C2. The estimatedmaximal values in fluorescence for WT and Ala108Ile were 0.221±0.009 and0.245±0.011 respectively, and were not significantly different (p>0.1).

Example 4 Stability of Factor VIII Arg121Cys/Leu2302Cys and Ala108IleVariants

The above results demonstrate that introduction of the disulfide bridgeor increasing the hydrophobic character at the A1-C2 interfacestabilizes this inter-domain interaction. To test the functionalconsequences of these mutations, stability parameters of factor VIII(factor VIIIa) were evaluated by several methods. Thermal denaturationexperiments were performed at 57° C. as described supra. Data shown inFIG. 6A were fitted to a single exponential decay curve using non-linearleast squares regression. WT factor VIII (circles) decayed to ˜40% theinitial activity level in 6-7 min at 57° C. On the other hand, theArg121Cys/Leu2302Cys variant (triangles) retained >40% activity up to 20min, whereas the Ala108Ile variant (squares) retained this level for >30min. Overall, decay rates for the Arg121Cys/Leu2302Cys and Ala108Ilevariants obtained by curve-fit were reduced by 3.1- and 4.2-fold,respectively, compared to the WT factor VIII value (see Table 1 below).

TABLE 1 Properties of Wild type Factor VIII and Variants Specific FactorVIII Factor VIIIa Activity Decay Rate IC₅₀ Decay Rate (%) (min⁻¹) (M)(min⁻¹) WT 100.0 ± 8.2  0.143 ± 0.003 (1.0)  0.814 ± 0.010 (1.00) 0.154± 0.006 (1.00) R121C/L2302C 86.4 ± 3.8 0.047 ± 0.001 (0.32) 0.826 ±0.005 (1.01) 0.132 ± 0.004 (0.86) A108I 73.7 ± 3.9 0.034 ± 0.002 (0.24)0.892 ± 0.008 (1.10) 0.119 ± 0.008 (0.77) Specific activity wasdetermined by factor Xa generation assay as described above andexpressed as a relative activity compared to WT value. Factor VIII decaydata at 57° C. as shown in FIG. 6A, and factor VIIIa spontaneous decaydata as shown in FIG. 6C were fitted to a single exponential decaycurve. Guanidinium denaturation data as shown in FIG. 6B were fitted toa linear response curve by non-linear least squares regression and IC₅₀values with standard deviations were obtained. Values in parentheses arerelative to the WT value.

In a complementary series of experiments, factor VIII stability wasexamined following a 2 h exposure to 0.6-1.2 M guanidinium (FIG. 6B). Asthe concentration of guanidinium increased, factor VIII activity wasreduced to near zero as an indication of denaturation. Using the rangeof linear response (˜0.6-1 M), data points were fitted by a linearequation and the IC₅₀ values were obtained (see Table 1). Factor VIIIactivity of the Ala108Ile variant was significantly more stable than WTshowing a ˜10% higher IC₅₀ values compared with WT (p<0.001), while theIC₅₀ determined for the Arg121Cys/Leu2302Cys variant was only slightlyincreased (−2% greater than WT, Table 1). Overall, the stability datafor the disulfide bridged variant suggested that the covalent bondbetween A1 and C2 subunits significantly increased factor VIII thermalstability while showing little stabilizing effect in the presence ofguanidinium. This result indicated that dissociation of factor VIIIheavy and light chains may be a prominent cause for activity loss atelevated temperature, but that chain dissociation may not represent aprimary mode for activity loss due to chemical denaturation.Alternatively, the Ala108Ile mutation demonstrated a more globalprotective effect in increasing factor VIII stability towards eitherthermal or chemical denaturation. Control experiments were performed todetermine whether there was any time-dependent change in activityfollowing the thermal or chemical denaturation step and return of factorVIII to either ambient temperature or dilution of denaturant,respectively. Factor VIII was assayed from 30 seconds to 1 hour, and nosignificant change in activity was observed.

Factor VIIIa activity is labile due to A2 subunit dissociation followingproteolytic activation (Fay et al., “Human Factor VIIIa SubunitStructure: Reconstitution of Factor VIIIa from the Isolated A1/A3-C1-C2Dimer and A2 Subunit,” J. Biol. Chem. 266:8957-8962 (1991); Lollar etal., “pH-dependent Denaturation of Thrombin-activated Porcine FactorVIII,” J. Biol. Chem. 265:1688-1692 (1990); Lollar et al., “CoagulantProperties of Hybrid Human/Porcine Factor VIII Molecules,” J. Biol.Chem. 267:23652-23657 (1992), each of which is hereby incorporated byreference in its entirety) To determine whether these mutations affectedfactor VIIIa decay, experiments were performed to assess rates of lossof factor VIIIa activity over time. As shown in FIG. 6C, reactionconditions employed resulted in the loss of ˜50% of WT factor VIIIaactivity at ˜6 min after thrombin activation, while ˜10% activityremained after 16 min. The observed factor VIIIa activity decay wasslightly reduced for both Arg121Cys/Leu2302Cys and Ala108Ile variantswhich showed ˜40% activity in 7-8 min and demonstrated decay rates thatwere 1.2- and 1.3-fold greater than WT factor VIII, respectively (seeTable 1). These results demonstrated only minor effects on theinter-subunit interactions involving A2 subunit following modificationof the A1 and C2 domain interface.

Example 5 Stability of Reconstituted Ala108Ile or WT A1 Subunit withA3C1C2 or A3C1 Subunits

The thermal stability for the A1/A3C1C2 dimer was assessed following itsreconstitution from isolated subunits. Purified WT A1 or Ala108Ile A1subunits was reconstituted with A3C1C2 subunit at 37° C. for 2 hrs andthe stability of the A1/A3C1C2 dimer at elevated temperature (55° C.)was measured by factor Xa generation assay following addition of A2subunit as described in Methods. As shown in FIG. 7A, factor VIIIaactivity reconstituted from WT-A1 decayed to ˜25% its original value at20 min (circles) while ˜80% of the original activity level remained forthe A1 subunit containing the Ala108Ile mutation (triangles). Theestimated decay rates for WT and the mutant factor VIIIa forms were0.066±0.005 and 0.014±0.001 min⁻¹, respectively, showing a 4.6-fold ratereduction for the variant compared to WT factor VIII.

Similar reconstitution experiments were performed using the A3C1 subunitderived from the C2 domain-deleted factor VIII variant. The rationalefor this experiment was that if the enhanced stability of the Ala108IleA1 were due to interaction with the C2 domain following reassociationwith A3C1C2, then use of the truncated A3C1 for reconstitution wouldabrogate the enhanced stability of the variant. Experiments performedwith a C2-domain deleted factor VIII, described in an earlier report(Wakabayashi et al., “Factor VIII Lacking the C2 Domain Retains CofactorActivity in vitro,” J. Biol. Chem. 285:25176-25184 (2010), which ishereby incorporated by reference in its entirety), showed that thisvariant was marked less stable than WT factor VIII at elevatedtemperatures and rates of decay needed to be monitored at a relativelylower temperature (52° C.). Under these conditions, the C2-domaindeleted factor VIII variant decayed ˜40-fold faster than WT factor VIII.For this reason, stability studies following factor VIII reconstitutionswith the A3C1 light chain were performed at 52° C. Furthermore, in anearlier study (Wakabayashi et al., “Generation of Enhanced StabilityFactor VIII Variants by Replacement of Charged Residues at the A2 DomainInterface,” Blood 112:2761-2769 (2008), which is hereby incorporated byreference in its entirety), it was demonstrated that factor VIIIstability measured over a range of temperatures from 52-60° C. yieldedsimilar relative rates of decay when comparing a given factor VIIIvariant to WT. Consistent with observations using the C2 domain-deletedfactor VIII, reconstitutions using either A1 form with A3C1 wereobserved to yield an overall faster decay (FIG. 7B), with ˜50% activityreduction at 4 min at 52° C., than results observed followingreconstitutions with the intact A3C1C2. The estimated decay rates for WTand the variant factor VIIIa forms were similar (0.166±0.001 and0.154±0.013 min⁻¹, respectively). That the observed increase in thermalstability of the Ala108Ile variant was also observed followingreconstitutions using purified components supports the conclusion thatthe enhanced stability of the variant as compared with WT derived fromimproved interaction(s) between A1 and A3C1C2 subunits and required thepresence of C2 subunit.

Discussion of Examples 1-5

The preceding Examples illustrate interactions at the interface betweenfactor VIII A1 and C2 domains following preparation of two factor VIIIvariants, Arg121Cys/Leu2302Cys factor VIII, which possesses a nascentdisulfide bond spanning these domains, and Ala108Ile factor VIII, whichhas a larger hydrophobic side chain to better fill the inter-domainspace. Several other mutations were prepared at this region in anattempt to create a disulfide bond or to increase hydrophobicity. Thesevariants yielded low specific activity values, possibly resulting fromunfavorable changes in conformation, and their characterization was notpursued further. However, both variants studied exhibited enhancedinter-A1-C2 domain affinity resulting in increases in the observedstability of the factor VIII variants, especially related to thermaldenaturation.

The intermediate resolution (˜4 Å) X-ray structure of factor VIII (Ngoet al., “Crystal Structure of Human Factor VIII: Implications for theFormation of the Factor IXa:Factor VIIIa Complex,” Structure 16:597-606(2008), which is hereby incorporated by reference in its entirety)predicts the close proximity of Arg121 and Leu2302 with 7.7 Å separatingCa atoms (PDB#3CDZ). This spatial separation suggested the potential tobridge this distance by a disulfide bond (˜4-6 Å) following replacementof these residues with Cys, and provided that the side chains were in anacceptable orientation. Results evaluating the Arg121Cys/Leu2302Cysfactor VIII protein by western blotting in the absence and presence ofdisulfide bond reduction showed high efficiency bridging (>90%)constituting experimental proof for the opposing orientation of sidechains of these two residues in factor VIII. In addition, the X-raystructure also showed that the A1-C2 junction adjacent to Ala108 is richin hydrophobic groups represented by the Cβ carbon of Ala108 from the Cδof Leu2302, the Cβ of Ala2328, or the Cγ of Gln2329 (see FIG. 3C). Thus,it was believed that extended alkyl groups of side chains larger thanthe methyl group of Ala might contribute to enhanced binding energy. Ofseveral variants prepared to this region, replacement of Ala108 with Ileyielded a variant possessing near WT-like specific activity.

Both Arg121Cys/Leu2302Cys and Ala108Ile variants exhibited superiorstability parameters as compared with the WT protein. For example, thethermal decay rates for the Arg121Cys/Leu2302Cys and Ala108Ile factorVIII variants were reduced by 3.1- and 4.2-fold, respectively, ascompared with WT. Further dissection of the interaction with theAla108Ile variant was obtained following reconstitution of the A1/A3C1C2dimer using WT and variant A1 subunits as well as the truncated A3C1subunit derived from the C2 domain-deleted factor VIII. Enhancedstability of the variant compared with WT was observed using nativeA3C1C2, but similar stability parameter values for variant and WT wereobserved even in the absence of the C2 domain. Taken together, theseobservations support the belief that modulating the A1-C2 interface,either through covalent bridging or increased hydrophobic interaction,appeared to make an important contribution to overall protein stability.

The primary cause for thermal decay of FVIII is attributed todissociation of the heavy and light chains (Ansong et al., “Factor VIIIA3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site,”Biochemistry 44:8850-8857 (2005), which is hereby incorporated byreference in its entirety). This result is supported by the presentstudy showing that bridging the factor VIII heavy chain and light chainvia a disulfide bond between A1 and C2 domains preferentially reducedthermal decay as compared with chemical denaturation. Thus, chemicaldenaturation appears to represent a more global effect on factor VIIIstructure and less specific for chain dissociation. It was reportedearlier that several residues at the A2-A3 interface (Tyr1792, Tyr1786and Asp666) possibly contributed to the binding energy only in theactive factor VIIIa form (Wakabayashi et al., “Identification ofResidues Contributing to A2 Domain-dependent Structural Stability inFactor VIII and Factor VIIIa,” J. Biol. Chem. 283:11645-11651 (2008),which is hereby incorporated by reference in its entirety). Thus,interactions between the A2 domain of the heavy chain and A3C1C2 domainsof the light chain may be minimal in the pro-cofactor. Based upon thatearlier report and the present study, it is believed that in the factorVIII heterodimer, the predominant sources for binding energy likelyderive from A1 interactions with both the A3 and C2 domains.

On the other hand, the instability of factor VIIIa results from weakelectrostatic interactions between the A2 subunit and the A1/A3C1C2dimer (Fay et al., “Human Factor VIIIa Subunit Structure: Reconstitutionof Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit,” J.Biol. Chem. 266:8957-8962 (1991); Lollar et al., “pH-dependentDenaturation of Thrombin-activated Porcine Factor VIII,” J. Biol. Chem.265:1688-1692 (1990), each of which is hereby incorporated by referencein its entirety) and its dissociation leads to dampening of factor Xaseactivity (Lollar et al., “Coagulant Properties of Hybrid Human/PorcineFactor VIII Molecules,” J. Biol. Chem. 267:23652-23657 (1992); Fay etal., “Model for the Factor VIIIa-dependent Decay of the Intrinsic FactorXase: Role of Subunit Dissociation and Factor IXa-catalyzedProteolysis,” J. Biol. Chem. 271:6027-6032 (1996), each of which ishereby incorporated by reference in its entirety). Several factor VIIIpoint mutations have been shown to facilitate the rate of dissociationof A2 relative to wild type (WT) and these residues localize to eitherthe A1-A2 domain interface (Pipe et al., “Mild Hemophilia A Caused byIncreased Rate of Factor VIII A2 Subunit Dissociation: Evidence forNonproteolytic Inactivation of Factor VIIIa in vivo,” Blood 93:176-183(1999); Pipe et al., “Hemophilia A Mutations Associated with1-stage/2-stage Activity Discrepancy Disrupt Protein-proteinInteractions within the Triplicated A Domains of Thrombin-activatedFactor VIIIa,” Blood 97:685-691 (2001), each of which is herebyincorporated by reference in its entirety) or the A1 and C2 domains(Hakeos et al., “Hemophilia A Mutations Within the Factor VIII A2-A3Subunit Interface Destabilize Factor VIIIa and Cause One-Stage/Two-StageActivity Discrepancy,” Thromb. Haemost 88: 781-787 (2002), which ishereby incorporated by reference in its entirety). In U.S. PatentApplication Publ. No. 20090118184 to Fay et al., which is herebyincorporated by reference in its entirety, it was demonstrated thatreplacing the charged residues Asp519, Glu665, and Glu1984 with Ala orVal yielded increased factor VIII stability and in particular enhancedretention of the A2 subunit in factor VIIIa. Interestingly, neither thesingle mutants nor combinations of these mutations yielded factor VIIIvariants that showed reductions in the rate of thermal decay of greaterthan 2.3-fold, whereas the variants examined in the present study showedthermal decay rate reductions of 3- to 4-fold. Thus, the magnitude ofstability enhancement observed for the A1-C2 interface variants appearssomewhat larger than for the A2 domain-mediated interactions. However,while these variants clearly showed superior factor VIII stability,results from this study indicated essentially little if any effect ofthe interactions involving the A1 and C2 domains in stabilizing thefactor VIIIa cofactor, suggesting no linkage of these sites with sitesinvolved in A2 subunit retention. For this reason, it is thecombinations of these A1-C2 domain stabilizing mutations with A1-A2 orA3-A2 domain stabilizing mutations that appear to be most desirable.

A1 domain residues 110-126 are in close contact to the C2 domain. Theseresidues contain a Ca²⁺ binding site predicted by Ala-scanningmutagenesis (Wakabayashi et al., “Residues 110-126 in the A1 Domain ofFactor VIII Contain a Ca²⁺ Binding Site Required for Cofactor Activity,”J. Biol. Chem. 279:12677-12684 (2004), which is hereby incorporated byreference in its entirety) and subsequently identified in the X-raycrystal structure (Shen et al., “The Tertiary Structure and DomainOrganization of Coagulation Factor VIII,” Blood 111:1240-1247 (2008);Ngo et al., “Crystal Structure of Human Factor VIII: Implications forthe Formation of the Factor IXa:Factor VIIIa Complex,” Structure16:597-606 (2008), each of which is hereby incorporated by reference inits entirety). Interestingly, preliminary experiments assessingchelation of Ca²⁺ (and/or Cu²⁺) in factor VIII by EGTA yielded dramaticlosses in activity of WT factor VIII while showing more minimal effectson the activity of Arg121Cys/Leu2302Cys and Ala108Ile variants. Withoutbeing bound by belief, it is believed the functional effects of Ca²⁺occupancy at 110-126 in factor VIII were replaced by enhancedstabilizing interactions between the A1 and C2 domains in the variants.

In conclusion, results from Examples 1-5 demonstrate that interactionsbetween the A1 and C2 domains of factor VIII contribute to the integrityof the protein, providing significant energy for stabilizing themulti-domain structure of factor VIII. Furthermore, observations forenhancing factor VIII stability, in particular by increasingnon-covalent, hydrophobic interactions at the A1-C2 domain interfacesuggests that these variants could potentially represent superiortherapeutics in the treatment of hemophilia.

Example 6 Combination of R121c/L2302C or Ala108Ile Substitution with Oneor More Substitutions at the A1-A2 or A2-A3 Domain Interfaces

Based on the improved thermal and/or chemical stability afforded by theA1-C2 variants, it was also determined whether the mutations at theA1-C2 interface can be combined with the mutation at one of the A1-A2 orA2-A3 interfaces to generate a factor VIII with even greater stability.Previously tested stable factor VIII mutants, as described in U.S.Patent Application Publ. No. 2009/0118184 to Fay et al., which is herebyincorporated by reference in its entirety, showed up to 2-fold increasesin thermal stability compared with WT factor VIII in any combination ofthe mutation with Asp519Ala, Asp519Val, Glu665Ala, Glu665Val,Glu1984Ala, and Glu1984Val (A1-A2 or A2-A3 domain mutants).

These single point mutations or the double mutation Asp519Val/Glu665Valwere combined with either R121C/L2302C or Ala108Ile in a B-domainlessfactor VIII cDNA using the procedures described in Example 1. Thesefactor VIII mutants were expressed and purified using the proceduresdescribed in Example 1. Specific activity values showed that most of themutants (10 out of 13) showed normal factor VIII activity values (>60%WT) as measured by factor Xa generation assay (see Table 2 below).Thermal stability values were determined as described above in Example4. Many of the resulting mutants exhibited >5 fold increase in thermalstability (10/13 mutants, FIG. 8A), with Ala108Ile/Glu665Val andAla108Ile/Asp519Val/Glu665Val being the most stable mutants (˜10 foldincrease relative to WT). Most of the mutants also showed 15-30%increases in IC₅₀ value (guanidinium experiment) compared with WT (FIG.8B). In addition, the high factor VIIIa stability of the A domainmutants was mostly preserved (mutants (11/13) showed 2-10 fold increasein factor VIIIa stability relative to WT, FIG. 8C). Collectively,modification at the A1-C2 contacting region by covalent attachment orincreasing hydrophobic interaction improved factor VIII stability andthese modifications can be combined with A2 domain interface mutationsto provide essentially additive effects compared with either type ofmutation alone.

TABLE 2 Specific Activity of Factor VIII Variants FVIII variantsActivity (%) FVIII variants Activity (%) WT 100 D519V/E665V 90.0Ala108Ile 73.7 121CL 86.4 A108I/D519A 77.7 121CL/D519A 60.7 A108I/D519V69.5 121CL/D519V 79.5 A108I/E665A 98.8 121CL/E665A 0 A108I/E665V 83.8121CL/E665V 22.2 A108I/E1984A 43.4 121CL/E1984A 63.4 A108I/E1984V 94.1121CL/E1984V 90.3 A108I/D519V/E665V 76.5 Activity was measured by FXageneration assay and expressed as relative values compared to WTactivity. The single letter code is used to designate amino acidresidues: I (Ile), E (Glu), D (Asp), A (Ala) and V (Val). The variant121CL represents R121C/L2302C.

Thrombin generation assays were performed as previously described(Wakabayashi et al., “Combining Mutations of Charged Residues at the A2Domain Interface Enhances Factor VIII Stability over Single PointMutations,” J. Thromb. Haemost. 7:438-444 (2009), which is herebyincorporated by reference in its entirety) to determine the effects ofcombining the Ala108Ile mutation with an A2 domain interface mutation.For this analysis, the Asp519Val/Glu665Val double mutation was employed.Results in FIG. 9 compare WT factor VIII with Ala108Ile,Asp519Val/Glu665Val and the combined Ala108Ile/Asp519Val/Glu665Valvariants. Both the Ala108Ile and the Asp519Val/Glu665Val variants showedimproved thrombin generation parameter values compared with WT (seeTable 3 below). In addition, the combined Ala108Ile/Asp519Val/Glu665Valmutation yielded somewhat greater thrombin peak values and endogenousthrombin potential (“ETP”, which is the area under the curve andrepresents total thrombin generated) than either individual variant.This indicates a positive effect in combining these mutations.

TABLE 3 Thrombin Generation Assay Parameter Values Latent Time Peak TimePeak Value ETP FVIII variants (min) (min) (nM) (nM · min) WT 7.59 ± 0.0916.1 ± 0.22  88.2 ± 6.60 1217 ± 34  (1.00) (1.00) (1.00) (1.00) A108I7.09 ± 0.41 12.7 ± 0.56 175.3 ± 17.0 1831 ± 183 (0.93) (0.79) (1.98)(1.50) D519V/E665V 8.19 ± 0.18 13.4 ± 0.16 203.4 ± 18.2 1939 ± 251(1.07) (0.83) (2.30) (1.59) A108I/D519V/ 7.05 ± 0.27 11.9 ± 0.31 222.8 ±10.7 1990 ± 122 E665V (0.92) (0.73) (2.52) (1.64) Thrombin generationassays in the presence of 0.5 nM factor VIII proteins, 0.5 pM rTF, and 4μM PSPCPE vesicles were performed and parameter values were calculated.Data represents the average values of triplicate samples. Values inparentheses are relative to the WT value. The single letter code is usedto designate amino acid residues: I (Ile), E (Glu), D (Asp), A (Ala) andV (Val).

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. An isolated nucleic acid molecule encoding arecombinant factor VIII, wherein said recombinant factor VIII comprisesone or more mutations at an interface of A1 and C2 domains ofrecombinant factor VIII that result in enhanced stability of factorVIII, wherein the one or more mutations comprise substitution of one ormore amino acid residues with an amino acid residue having a higherhydrophobicity, and wherein the A1 domain of the recombinant factor VIIIcomprises a C2 domain interface having the amino acid sequence of KXS(SEQ ID NO: 19) and the substitution at the second residue is Valine,Isoleucine, or Leucine.
 2. The isolated nucleic acid molecule accordingto claim 1, wherein the recombinant factor VIII further comprises one ormore of (i) factor IXa and/or factor X binding domains modified toenhance the affinity of the recombinant factor VIII for one or both offactor IXa and factor X; (ii) modified sites that enhance secretion inculture; (iii) modified serum protein binding sites that enhance thecirculating half-life thereof; (iv) at least one glycosylationrecognition sequence that is effective in decreasing antigenicity and/orimmunogenicity thereof; (v) a modified A1 domain calcium-binding sitethat improves specific activity of the recombinant factor VIIIa; (vi) amodified activated protein C-cleavage site; (vii) a modified A1 and A2domain interface; and (viii) a modified A2 and A3 domain interface. 3.The isolated nucleic acid molecule according to claim 1, wherein therecombinant factor VIII further comprises a modified A1 domaincalcium-binding site that improves specific activity of the recombinantfactor VIIIa.
 4. The isolated nucleic acid molecule according to claim3, wherein the modified A1 domain calcium-binding site comprises aGlu→Ala substitution corresponding to position 113 of SEQ ID NO:
 2. 5.The isolated nucleic acid molecule according to claim 1, wherein therecombinant factor VIII further comprises one or more mutations at theA1 and A2 domain interface and/or one or more mutations at the A2 and A3domain interface.
 6. The isolated nucleic acid molecule according toclaim 5, wherein the one or more mutations comprise substitution of oneor more charged amino acid residues with a hydrophobic amino acidresidue at the A1 and A2 domain interface and/or A2 and A3 domaininterface.
 7. The isolated nucleic acid molecule according to claim 6,wherein the charged amino acid residue at the A1 and A2 domain interfaceand/or A2 and A3 domain interface is either Glu or Asp, and thehydrophobic amino acid substitution at the A1 and A2 domain interfaceand/or A2 and A3 domain interface is one of Ala, Val, Ile, Leu, Met,Phe, or Trp.
 8. The isolated nucleic acid molecule according to claim 6,wherein the one or more mutations comprise a substitution of a Glu287residue of wild type factor VIII, a substitution of an Asp302 residue ofwild type factor VIII, a substitution of an Asp519 residue of wild typefactor VIII, a substitution of a Glu665 residue of wild type factorVIII, a substitution of a Glu1984 residue of wild type factor VIII, orcombinations thereof.
 9. The isolated nucleic acid molecule according toclaim 8, wherein the one or more mutations comprise: (i) an Asp→Alasubstitution at a position corresponding to residue 302 of SEQ ID NO: 2;(ii) a Glu→Ala substitution at a position corresponding to residue 287of SEQ ID NO: 2; (iii) a Glu→Ala or Glu→Val substitution at a positioncorresponding to residue 665 of SEQ ID NO: 2; (iv) a Asp→Ala or Asp→Valsubstitution at a position corresponding to residue 519 of SEQ ID NO: 2;(v) a Glu→Ala or Glu→Val substitution at a position corresponding toresidue 1984 of SEQ ID NO: 2; or (vi) combinations of any two or more ofthe substitutions (i)-(v).
 10. The isolated nucleic acid moleculeaccording to claim 1, wherein the nucleic acid is RNA.
 11. The isolatednucleic acid molecule according to claim 1, wherein the nucleic acid isDNA.
 12. A recombinant expression system comprising a DNA molecule ofclaim
 11. 13. The recombinant expression system according to claim 12,wherein the recombinant expression system is a viral vector.
 14. Therecombinant expression system according to claim 13, wherein the viralvector is an adeno-associated viral vector.
 15. A recombinant host cellcomprising the isolated nucleic acid molecule according to claim
 1. 16.The recombinant host cell according to claim 15, wherein the recombinanthost cell is a dermal fibroblast.
 17. An implantable device comprising aplurality of the recombinant host cells according to claim 15 containedwithin the device.
 18. A recombinant host cell comprising a DNA moleculeof claim
 11. 19. An implantable device comprising a plurality of therecombinant host cells according to claim 18 contained within thedevice.
 20. A method of treating an animal for hemophilia A, said methodcomprising: administering to an animal exhibiting hemophilia A aneffective amount of the nucleic acid molecule of claim 1, whereby theanimal expresses the recombinant factor VIII and exhibits effectiveblood clotting following vascular injury.
 21. The method according toclaim 20, wherein said animal is a mammal.
 22. The method according toclaim 21, wherein said mammal is selected from the group consisting ofhuman, rat, mouse, guinea pig, dog, cat, monkey, chimpanzee, orangutan,cow, horse, sheep, pig, goat, rabbit, and chicken.
 23. The methodaccording to claim 20, wherein said nucleic acid molecule comprises aviral vector.
 24. The method according to claim 23, wherein said viralvector is an adeno-associated viral vector.
 25. A method of treating ananimal for hemophilia A, said method comprising: administering to ananimal exhibiting hemophilia A a recombinant host cell of claim 15,whereby the animal expresses the recombinant factor VIII and exhibitseffective blood clotting following vascular injury.
 26. The methodaccording to claim 25, wherein said administering comprises implantinginto the animal a device containing a plurality of said recombinant hostcells.
 27. The method according to claim 25, wherein the recombinanthost cell is a dermal fibroblast.
 28. The method according to claim 25,wherein said animal is a mammal.
 29. The method according to claim 28,wherein said mammal is selected from the group consisting of human, rat,mouse, guinea pig, dog, cat, monkey, chimpanzee, orangutan, cow, horse,sheep, pig, goat, rabbit, and chicken.